Human antibody drug conjugates against tissue factor

ABSTRACT

Antibody drug conjugates against tissue factor. Also disclosed are pharmaceutical compositions comprising the antibodies and antibody drug conjugates, and therapeutic and diagnostic methods for using the antibodies and antibody drug conjugates.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/EP2011/059917 filed Jun. 15, 2011,which claims priority to 61/354,970 filed Jun. 15, 2010; PA 2010 00529filed Jun. 15, 2010; 61/434,776 filed Jan. 20, 2011; and PA 2011 00039filed Jan. 20, 2011. The contents of the aforementioned applications arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to antibody drug conjugates (ADCs), wherethe antibodies bind an epitope on tissue factor. Such ADCs are inparticular useful in the treatment of cancer, inflammation and vasculardiseases.

BACKGROUND OF THE INVENTION

Tissue factor (TF), also called thromboplastin, factor III or CD142 is aprotein present in subendothelial tissue, platelets, and leukocytesnecessary for the initiation of thrombin formation from the zymogenprothrombin. Thrombin formation ultimately leads to the coagulation ofblood. Tissue factor enables cells to initiate the blood coagulationcascades, and it functions as the high-affinity receptor for thecoagulation factor VII (FVII), a serine protease. The resulting complexprovides a catalytic event that is responsible for initiation of thecoagulation protease cascades by specific limited proteolysis. Unlikethe other cofactors of these protease cascades, which circulate asnonfunctional precursors, this factor is a potent initiator that isfully functional when expressed on cell surfaces.

Tissue factor is the cell surface receptor for the serine proteasefactor VIIa (FVIIa). Binding of FVIIa to tissue factor starts signalingprocesses inside the cell, said signaling function playing a role inangiogenesis. Whereas angiogenesis is a normal process in growth anddevelopment, as well as in wound healing, it is also a fundamental stepin the transition of tumors from a dormant state to a malignant state:when cancer cells gain the ability to produce proteins that participatein angiogenesis, so called angiogenic growth factors, these proteins arereleased by the tumor into nearby tissues, and stimulate new bloodvessels to sprout from existing healthy blood vessels toward and intothe tumor. Once new blood vessels enter the tumor, it can rapidly expandits size and invade local tissue and organs. Through the new bloodvessels, cancer cells may further escape into the circulation and lodgein other organs to form new tumors (metastases).

Further, TF plays a role in inflammation. The role of TF is assumed tobe mediated by blood coagulation (A. J. Chu: “Tissue factor mediatesinflammation” in Archives of biochemistry and biophysics, 2005, vol.440, No. 2, pp. 123-132). Accordingly, the inhibition of TF, e.g. by amonoclonal anti-TF antibody is of significance in interrupting thecoagulation-inflammation cycle in contribution to not onlyanti-inflammation but also to vascular diseases.

TF expression is observed in many types of cancer and is associated withmore aggressive disease. Furthermore, human TF also exists in a solublealternatively-spliced form, asHTF. It has recently been found that asHTFpromotes tumor growth (Hobbs et al., 2007 Thrombosis Res. 120(2)S13-S21).

Although much progress has been made, there remains a need for improvedmethods of treating serious diseases, e.g. improved treatment of cancer,inflammation and vascular disease based on therapeutic antibodies.

It is accordingly an object of the present invention to provide highlyspecific and effective anti-TF antibody drug conjugates, in particularfor the use in the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention relates to novel anti-TF antibody drug conjugateswhich are useful for the treatment of cancer, inflammation and vasculardiseases. The anti-TF antibody drug conjugates of the present inventionare highly effective in killing cells expressing tissue factor (TF).Furthermore, the anti-TF antibody drug conjugates are advantageous byhaving limited or no inhibition of coagulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of sequences of the antibodies of the presentinvention.

SEQ ID NOs is listed in parentheses to the right of the sequence.

CDR1, CDR2 and CDR3 according to Kabat are indicated as follows:sequences in italics and bold represent the CDR1 region, underlinedsequences represent the CDR2 region, bold sequences represent the CDR3region.

FIG. 2: IgG4 sequences (SEQ ID NO: 81-82)

SEQ ID NO: 81: The amino acid sequence of the wild-type CH region ofhuman IgG4. Sequences in italics represent the CH1 region, highlightedsequences represent the hinge region, regular sequences represent theCH2 region and underlined sequences represent the CH3 region.

SEQ ID NO: 82: The amino acid sequence of the hingeless CH region of ahuman IgG4

FIG. 3: Binding of anti-TF HuMabs to the extracellular domain of TF.Binding was determined by ELISA. EC₅₀ values are the mean of 3experiments.

FIG. 4: Binding of anti-TF HuMabs to membrane-bound TF on MDA-MD-231cells. Binding was determined by FACS analysis and the antibodies weresplit into three groups shown in a), b) and c), see also WO 10/066,803where antibodies were split into cross-block groups.

FIG. 5: Inhibition of FVIIa binding by anti-TF HuMabs, was measured byFACS analysis. Data shown are mean fluorescence intensities (MFI) forFVIIa binding in the presence of increasing concentrations of anti-TFHuMabs. MFI for 100 nM FVIIa in the absence of anti-TF HuMabs was149,942. One representative experiment is shown.

FIG. 6: Dose-dependent induction of cell killing byanti-kappa-ETA′-conjugated anti-TF HuMabs.

FIG. 7: Binding of anti-TF HuMabs and ADCs to recombinant protein of theTF extracellular domain, determined by ELISA. One representativeexperiment is shown.

FIG. 8: In vitro dose-dependent induction of cell killing by anti-TFADCs. One representative experiment is shown for each cell line: A431(a), HPAF-II (b) and NCI-H441 (c). Data shown are percentages survival±S.E.M. of duplicate wells of cells treated with anti-TF ADCs.

FIG. 9: In vivo efficacy of anti-TF ADCs in therapeutic treatment ofA431 and HPAF-II xenografts in SCID mice. Mice with established A431 (A)or HPAF-II (B) tumors were treated with anti-TF ADCs, Data shown aremean tumor volumes ±S.E.M. per group (n=7 mice per group).

FIG. 10: SDS-PAGE analysis of ADCs and unconjugated IgG1 to teststability. Samples were analyzed by SDS-PAGE at the start of the study(t=0) (a-d) or after storage at 5° C. and <−65° C. for three months(e-h), (a, c) lane 1, 9: molecular weight (MW) marker, lane 2: IgG1internal control, lane 3: HuMab-TF-098, lane 4: HuMab-TF-098-vcMMAE,lane 5: HuMab-TF-098-mcMMAF, lane 6: HuMab-TF-011, lane 7:HuMab-TF-011-vcMMAE, lane 8: HuMab-TF-mcMMAF, (b, d) lane 1, 9, 10: MWmarker, lane 2: IgG1 internal control, lane 3: HuMab-TF-111, lane 4:HuMab-TF-111-vcMMAE, lane 5: HuMab-TF-111-mcMMAF, lane 6: IgG1-b12, lane7: IgG1-b12-vcMMAE, lane 8: IgG1-b12-mcMMAF.

(e, g) lane 1, 11: MW marker, lane 2: IgG1 internal control, lane 3:HuMab-TF-098-vcMMAE after 3 months at <−65° C., lane 4:HuMab-TF-098-vcMMAE after 3 months at 5° C., lane 5: HuMab-TF-098-mcMMAFafter 3 months at <−65° C., lane 6: HuMab-TF-098-mcMMAF after 3 monthsat 5° C., lane 7: HuMab-TF-011-vcMMAE after 3 months at <−65° C., lane8: HuMab-TF-011-vcMMAE after 3 months at 5° C., lane 9:HuMab-TF-011-mcMMAF after 3 months at <−65° C., lane 10:HuMab-TF-011-mcMMAF after 3 months at 5° C. (1, h) lane 1, 11, 12: MWmarker, lane 2: IgG1 internal control, lane 3: HuMab-TF-111-vcMMAE after3 months at <−65° C., lane 4: HuMab-TF-111-vcMMAE after 3 months at 5°C., lane 5: HuMab-TF-111-mcMMAF after 3 months at <−65° C., lane 6:HuMab-TF-111-mcMMAF after 3 months at 5° C., lane 7: IgG1-b12-vcMMAEafter 3 months at <−65° C., lane 8: IgG1-b12-vcMMAE after three monthsat 5° C., lane 9: IgG1-b12-mcMMAF after 3 months at <−65° C., lane 10:IgG1-b12-mcMMAF after 3 months at 5° C.

For non-reducing conditions, sizes of different heavy chain (H) lightchain (L) combinations are indicated: 148 kDa (HHLL), 125 kDa (HHL), 99kDa (HH), 67 kDa (HL), 51 kDa (H) and 25 kDa (L).

FIG. 11: High Performance Size Exclusion Chromatography (HP-SEC)profiles of HuMab-TF-098-vcMMAE (a), HuMab-TF-098-mcMMAF (b),HuMab-TF-011-vcMMAE (c), HuMab-TF-011-mcMMAF (d), HuMab-TF-111-vcMMAE(e), HuMab-TF-111-mcMMAF (1), IgG1-b12-vcMMAE (g) and IgG1-b12-mcMMAF(h) at the start of the study and after storage at <−65° C. or 5° C. forthree months.

FIG. 12: Binding assay of ADCs and unconjugated IgG1 to TF-ECDHis totest stability, Samples were analyzed for binding at the start of thestudy (t=0) or after storage at 5° C. and <−65° C. for three months.

FIG. 13: In vivo dose-response of anti-TF ADCs in therapeutic treatmentof HPAF-II xenografts in SCID mice. Mice with established HPAF-II tumorswere treated with anti-TF vcMMAE ADCs. Data shown are mean tumor volumes±S.E.M. per group (n=8 mice per group).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “tissue factor”, “TF”, “CD142”, “tissue factor antigen”, “TFantigen” and “CD142 antigen” are used interchangeably herein, and,unless specified otherwise, include any variants, isoforms and specieshomologs of human tissue factor which are naturally expressed by cellsor are expressed on cells transfected with the tissue factor gene.Tissue factor may be the sequence Genbank accession NP_(—)001984 used inexample 1.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)), Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H) or VH) and a heavychain constant region (C_(H) or CH). The heavy chain constant regiontypically is comprised of three domains, C_(H)1, C_(H)2, and C_(H)3.Each light chain typically is comprised of a light chain variable region(abbreviated herein as V_(L) or VL) and a light chain constant region(C_(L) or CL). The light chain constant region typically is comprised ofone domain, C_(L). The V_(H) and V_(L) regions may be further subdividedinto regions of hypervariability (or hypervariable regions, which may behypervariable in sequence and/or form of structurally defined loops),also termed complementarity-determining regions (CDRs), interspersedwith regions that are more conserved, termed framework regions (FRs).Each V_(H) and V_(L) is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.Biol. 196, 901-917 (1987)). Typically, the numbering of amino acidresidues in this region is performed by the method described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed, PublicHealth Service, National Institutes of Health, Bethesda, Md., (1991)(phrases such as variable domain residue numbering as in Kabat oraccording to Kabat herein refer to this numbering system for heavy chainvariable domains or light chain variable domains). Using this numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of V_(H) CDR2 and insertedresidues (for instance residues 82a, 82b, and 82c, etc, according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with a halflife of significant periods of time, such as at least about 30 minutes,at least about 45 minutes, at least about one hour, at least about twohours, at least about four hours, at least about 8 hours, at least about12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5,6, 7 or more days, etc., or any other relevant functionally-definedperiod (such as a time sufficient to induce, promote, enhance, and/ormodulate a physiological response associated with antibody binding tothe antigen and/or time sufficient for the antibody to recruit aneffector activity). The variable regions of the heavy and light chainsof the immunoglobulin molecule contain a binding domain that interactswith an antigen. The constant regions of the antibodies (Abs) maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (such as effector cells)and components of the complement system such as C1q, the first componentin the classical pathway of complement activation. As indicated above,the term antibody herein, unless otherwise stated or clearlycontradicted by context, includes fragments of an antibody that retainthe ability to specifically bind to the antigen. It has been shown thatthe antigen-binding function of an antibody may be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antibody” include (i) a Fab′ or Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains, or a monovalent antibody as described inWO2007059782 (Genmab A/S); (ii) F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting essentially of the V_(H) andC_(H)1 domains; (iv) a Fv fragment consisting essentially of the V_(L)and V_(H) domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., Nature 341, 544-546 (1989)), which consists essentially ofa V_(H) domain and also called domain antibodies (Holt et al; TrendsBiotechnol, 2003 November; 21(11):484-90); (vi) camelid or nanobodies(Revets et al; Expert Opin Biol Ther, 2005 January; 5(1):111-24) and(vii) an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they may be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain antibodies or single chain Fv (scFv),see for instance Bird et al., Science 242, 423-426 (1988) and Huston etal, PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies areencompassed within the term antibody unless otherwise noted or clearlyindicated by context. Although such fragments are generally includedwithin the meaning of antibody, they collectively and each independentlyare unique features of the present invention, exhibiting differentbiological properties and utility. These and other useful antibodyfragments in the context of the present invention are discussed furtherherein. It also should be understood that the term antibody, unlessspecified otherwise, also includes polyclonal antibodies, monoclonalantibodies (mAbs), antibody-like polypeptides, such as chimericantibodies and humanized antibodies, and antibody fragments retainingthe ability to specifically bind to the antigen (antigen-bindingfragments) provided by any known technique, such as enzymatic cleavage,peptide synthesis, and recombinant techniques. An antibody as generatedcan possess any isotype.

In the context of the present invention the term “ADC” refers to anantibody drug conjugate, which in the context of the present inventionrefers to an anti-TF antibody, which is coupled to another moiety asdescribed in the present application.

An “anti-TF antibody” is an antibody as described above, which bindsspecifically to the antigen tissue factor or tissue factor antigen.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

In a preferred embodiment, the antibody of the antibody drug conjugate,or the antibody drug conjugate of the invention is isolated. An“isolated antibody” or “isolated antibody drug conjugate” as usedherein, is intended to refer to an antibody or antibody drug conjugatewhich is substantially free of other antibodies having differentantigenic specificities (for instance an isolated antibody thatspecifically binds to tissue factor is substantially free of antibodiesthat specifically bind antigens other than tissue factor). An isolatedantibody drug conjugate as used herein, is intended to refer to anantibody drug conjugate which is also substantially free of “freetoxin”, wherein “free toxin” is intended to mean toxin which is notconjugated to the antibody. The term “substantially free of” as used inrelation to the toxin may in particular mean that less than 5%, such asless than 4%, or less than 3%, or less than 2%, or less than 1.5%, orless than 1%, or less than 0.5% unconjugated drug is present whendetermined as described in Example 16. An isolated antibody or isolatedantibody drug conjugate that specifically binds to an epitope, isoformor variant of human tissue factor may, however, have cross-reactivity toother related antigens, for instance from other species (such as tissuefactor species homologs). Moreover, an isolated antibody or antibodydrug conjugate may be substantially free of other cellular materialand/or chemicals. In one embodiment of the present invention, two ormore “isolated” monoclonal antibodies or antibody drug conjugates havingdifferent antigen-binding specificities are combined in a well-definedcomposition.

When used herein in the context of two or more antibodies, the term“competes with” or “cross-competes with” indicates that the two or moreantibodies compete for binding to TF, e.g. compete for TF binding in theassay as described in Example 12 of WO 10/066,803. For some pairs ofantibodies, competition as in the assay of Example 12 of WO 10/066,803is only observed when one antibody is coated on the plate and the otheris used to compete, and not vice versa. The term “competes with” whenused herein is also intended to cover such combinations of antibodies.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. The monoclonal antibody or composition thereofmay be drug conjugated antibodies according to the present invention. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to antibodies displaying a single bindingspecificity which have variable and constant regions derived from humangermline immunoglobulin sequences. The human monoclonal antibodies maybe generated by a hybridoma which includes a B cell obtained from atransgenic or transchromosomal non-human animal, such as a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene, fused to an immortalized cell.

As used herein, the terms “binding” or “specifically binds” in thecontext of the binding of an antibody to a pre-determined antigentypically is a binding with an affinity corresponding to a K_(D) ofabout 10⁻⁷ M or less, such as about 10⁻⁸ or less, such as about 10⁻⁹ Mor less, about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or even less whendetermined by for instance surface plasmon resonance (SPR) technology ina BiAcore 3000 instrument using the antigen as the ligand and theantibody as the analyte, and binds to the predetermined antigen with anaffinity corresponding to a K_(D) that is at least ten-fold lower, suchas at least 100 fold lower, for instance at least 1,000 fold lower, suchas at least 10,000 fold lower, for instance at least 100,000 fold lowerthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the pre-determined antigen or a closely-relatedantigen. The amount with which the affinity is lower is dependent on theK_(D) of the antibody, so that when the K_(D) of the antibody is verylow (that is, the antibody is highly specific), then the amount withwhich the affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000 fold.

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, refers to the associationrate constant of a particular antibody-antigen interaction.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.

The term “K_(A)” (M⁻¹), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the k_(a) by the k_(d).

As used herein, the term “internalization”, when used in the context ofa TF antibody includes any mechanism by which the antibody isinternalized into a TF-expressing cell from the cell-surface. Theinternalization of an antibody can be evaluated in an indirect or directassay where the effect of an internalized antibody-toxin conjugate orcomplex is measured (such as, e.g., the anti-kappa-ETA′assay of Example15 or the internalization and cell killing assay of Example 18).Generally, a direct assay is used for measuring internalization ofantibody drug conjugates, such as the assay described in Example 18herein, while indirect assays may be used for measuring internalizationof antibodies which are then pre-incubated with a secondary conjugatedantibody, such as the assay described in Example 15 herein.

The present invention also provides, in one embodiment, antibodiescomprising functional variants of the V_(L) region, V_(H) region, or oneor more CDRs of the antibodies of the examples. A functional variant ofa V_(L), V_(H), or CDR used in the context of an anti-TF antibody stillallows the antibody to retain at least a substantial proportion (atleast about 50%, 60%, 70%, 80%, 90%, 95% or more) of theaffinity/avidity and/or the specificity/selectivity of the parentantibody and in some cases such an anti-TF antibody may be associatedwith greater affinity, selectivity and/or specificity than the parentantibody.

Such functional variants typically retain significant sequence identityto the parent antibody. The percent identity between two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences may be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences may be determinedusing the GAP program in the GCG software package (available atwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percentidentity between two nucleotide or amino acid sequences may also bedetermined using the algorithm of E. Meyers and W. Miller, Comput. Appl.Biosi 4, 11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences may be determined using the Needlemanand Wunsch, J. Mol. Biol. 48, 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

The sequence of CDR variants may differ from the sequence of the CDR ofthe parent antibody sequences through mostly conservative substitutions;for instance at least about 35%, about 50% or more, about 60% or more,about 70% or more, about 75% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more (e.g., about 65-99%, such asabout 96%, 97% or 98%) of the substitutions in the variant areconservative amino acid residue replacements.

The sequence of CDR variants may differ from the sequence of the CDR ofthe parent antibody sequences through mostly conservative substitutions;for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1of the substitutions in the variant are conservative amino acid residuereplacements.

In the context of the present invention, conservative substitutions maybe defined by substitutions within the classes of amino acids reflectedin one or more of the following three tables:

Amino Add Residue Classes for Conservative Substitutions

Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), andHis (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), andGln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L),and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

Alternative Conservative Amino Acid Residue Substitution Classes

1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turnformation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q,T, K, S, G, P, D, E, and R

More conservative substitution groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W.H. Freeman and Company.

In one embodiment of the present invention, conservation in terms ofhydropathic/hydrophilic properties and residue weight/size also issubstantially retained in a variant CDR as compared to a CDR of anantibody of the examples (e.g., the weight class, hydropathic score, orboth of the sequences are at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or more (e.g., about65-99%) retained). For example, conservative residue substitutions mayalso or alternatively be based on the replacement of strong or weakbased weight based conservation groups, which are known in the art.

The retention of similar residues may also or alternatively be measuredby a similarity score, as determined by use of a BLAST program (e.g.,BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62,Open Gap=11 and Extended Gap=1). Suitable variants typically exhibit atleast about 45%, such as at least about 55%, at least about 65%, atleast about 75%, at least about 85%, at least about 90%, at least about95%, or more (e.g., about 70-99%) similarity to the parent peptide.

As used herein, “isotype” refers to the immunoglobulin class (forinstance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specifically antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specifically antigen binding peptide).

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody isat least 90%, such as at least 95%, for instance at least 96%, such asat least 97%, for instance at least 98%, or such as at least 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline immunoglobulin gene. Typically, outside the heavy chainCDR3, a human antibody derived from a particular human germline sequencewill display no more than 20 amino acid differences, e.g. no more than10 amino acid differences, such as no more than 9, 8, 7, 6 or 5, forinstance no more than 4, 3, 7, or 1 amino acid difference from the aminoacid sequence encoded by the germline immunoglobulin gene.

As used herein, the term “inhibits growth” (e.g. referring to cells,such as tumor cells) is intended to include any measurable decrease inthe cell growth when contacted with an anti-TF antibody drug conjugateas compared to the growth of the same cells not in contact with ananti-TF antibody drug conjugate, e.g., the inhibition of growth of acell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 99%, or 100%. Such a decrease in cell growth can occur by a varietyof mechanisms mechanisms exerted by the anti-TF antibody and drug,either individually or in combination, e.g., antibody-dependentcell-mediated phagocytosis (ADCP), antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and/orapoptosis, or G2/M cell cycle arrest and apoptosis such as may beinduced by an interaction of the auristatin with tubulin.

The term “stabilized IgG4 antibody” refers to an IgG4 antibody which hasbeen modified to reduce half-molecule exchange (see WO 2008/145142(Genmab A/S) or van der Neut Kolfschoten M et al., (2007) Science 14;317(5844) and references therein).

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, forinstance lymphocytes (such as B cells and T cells including cytolytic Tcells (CTLs)), killer cells, natural killer cells, macrophages,monocytes, eosinophils, polymorphonuclear cells, such as neutrophils,granulocytes, mast cells, and basophils. Some effector cells expressspecific Fc receptors (FcRs) and carry out specific immune functions. Insome embodiments, an effector cell is capable of inducing ADCC, such asa natural killer cell, capable of inducing ADCC. For example, monocytes,macrophages, which express FcRs are involved in specific killing oftarget cells and presenting antigens to other components of the immunesystem, or binding to cells that present antigens. In some embodiments,an effector cell may phagocytose a target antigen or target cell. Theexpression of a particular FcR on an effector cell may be regulated byhumoral factors such as cytokines. For example, expression of FcγRI hasbeen found to be up-regulated by interferon γ (IFN-γ) and/or granulocytecolony stimulating factor (G-CSF). This enhanced expression increasesthe cytotoxic activity of FcγRI-bearing cells against target cells. Aneffector cell can phagocytose or lyse a target antigen or a target cell.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double-stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (for instance bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (such asnon-episomal mammalian vectors) may be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the present invention is intended toinclude such other forms of expression vectors, such as viral vectors(such as replication-defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced. It should be understood that such terms areintended Cu refer not only to the particular subject cell, but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing the antibody, such as CHO cells, NS/0 cells,HEK293 cells, plant cells, or fungi, including yeast cells.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces human anti-TFantibodies when immunized with TF antigen and/or cells expressing TF.The human heavy chain transgene may be integrated into the chromosomalDNA of the mouse, as is the case for transgenic mice, for instance HuMAbmice, such as HCo7, HCo17, HCo20 or HCo12 mice, or the human heavy chaintransgene may be maintained extrachromosomally, as is the case fortranschromosomal KM mice as described in WO02/43478. Such transgenic andtranschromosomal mice (collectively referred to herein as “transgenicmice”) are capable of producing multiple isotypes of human monoclonalantibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) byundergoing V-D-J recombination and isotype switching, Transgenic,nonhuman animal can also be used for production of antibodies against aspecific antigen by introducing genes encoding such specific antibody,for example by operatively linking the genes to a gene which isexpressed in the milk of the animal.

“Treatment” refers to the administration of an effective amount of atherapeutically active compound of the present invention with thepurpose of easing, ameliorating, arresting or eradicating (curing)symptoms or disease states.

An “effective amount” or “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. A therapeutically effective amountof an anti-TF antibody drug conjugate may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the anti-TF antibody drug conjugate to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody orantibody portion are outweighed by the therapeutically beneficialeffects.

An “anti-idiotypic” (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody.

FURTHER ASPECTS AND EMBODIMENTS OF THE INVENTION

The invention provides an anti-TF antibody drug conjugate.

In one aspect the invention provides an antibody drug conjugatecomprising an antibody which binds to tissue factor and which comprises

(i) a VH region comprising a CDR1 region having the amino acid sequenceset forth in SEQ ID NO:6, a CDR2 region having the amino acid sequenceset forth in SEQ ID NO: 7, and a CDR3 region having region having theamino acid sequence set forth in SEQ ID NO: 8, and a VL regioncomprising a CDR1 region having the amino acid sequence set forth in SEQID NO:46, a CDR2 region having the amino acid sequence set forth in SEQID NO: 47, and a CDR3 region having region having the amino acidsequence set forth in SEQ ID NO: 48, or(ii) a VH region comprising a CDR1 region having the amino acid sequenceset forth in SEQ ID NO:34, a CDR2 region having the amino acid sequenceset forth in SEQ ID NO: 35, and a CDR3 region having region having theamino acid sequence set forth in SEQ ID NO: 36, and a VL regioncomprising a CDR1 region having the amino acid sequence set forth in SEQID NO:74, a CDR2 region having the amino acid sequence set forth in SEQID NO; 75, and a CDR3 region having region having the amino acidsequence set forth in SEQ ID NO: 76, or(iii) a VH region comprising a CDR1 region having the amino acidsequence set forth in SEQ ID NO:38, a CDR2 region having the amino acidsequence set forth in SEQ ID NO: 39, and a CDR3 region having regionhaving the amino acid sequence set forth in SEQ ID NO: 40, and a VLregion comprising a CDR1 region having the amino acid sequence set forthin SEQ ID NO:78, a CDR2 region having the amino acid sequence set forthin SEQ ID NO: 79, and a CDR3 region having region having the amino acidsequence set forth in SEQ ID NO: 80, or(iv) a VH region comprising a CDR1 region having the amino acid sequenceset forth in SEQ ID NO:2, a CDR2 region having the amino acid sequenceset forth in SEQ ID NO: 3, and a CDR3 region having region having theamino acid sequence set forth in SEQ ID NO: 4, and a VL regioncomprising a CDR1 region having the amino acid sequence set forth in SEQID NO: 42, a CDR2 region having the amino acid sequence set forth in SEQID NO: 43, and a CDR3 region having region having the amino acidsequence set forth in SEQ ID NO: 44, or(v) a variant of any of said antibodies, wherein said variant preferablyhas at most 1, 2 or 3 amino-acid modifications, more preferablyamino-acid substitutions, such as conservative amino-acid substitutionsin said sequences,wherein the antibody has been conjugated to an auristatin or afunctional peptide analog or derivate thereof via a linker.

In one embodiment the antibody comprises

(i) a VH region comprising an amino acid sequence of SEQ ID NO: 5 and aVL region comprising an amino acid sequence of SEQ ID NO: 45, or

(ii) a VH region comprising an amino acid sequence of SEQ ID NO: 33 anda VL region comprising an amino acid sequence of SEQ ID NO: 73, or

(iii) a VH region comprising an amino acid sequence of SEQ ID NO: 37 anda \/L region comprising an amino acid sequence of SEQ ID NO: 77, or

(iv) a VH region comprising an amino acid sequence of SEQ ID NO: 1 and aVL region comprising an amino acid sequence of SEQ ID NO: 41.

In one embodiment the antibody is a full length antibody.

In one embodiment the antibody is a fully human monoclonal IgG1antibody, such as an IgG1, κ. In another embodiment the antibody is afully human monoclonal stabilized IgG4 antibody.

In one embodiment the auristatin is monomethyl auristatin E (MMAE):

wherein the wavy line indicates the attachment: site for the linker.

In one embodiment the auristatin is monomethyl auristatin F (MMAF):

wherein the wavy line indicates the attachment site for the linker.

In one embodiment the linker is attached to sulphydryl residues of theanti-TF antibody obtained by (partial) reduction of the anti-TFantibody.

In one embodiment the linker-auristatin is MC-vc-PAB-MMAF (alsodesignated as vcMMAF) or MC-vc-PAB-MMAE (also designated as vcMMAE):

wherein p denotes a number of from 1 to 8, e.g. p may be from 3-5, Srepresents a sulphydryl residue of the anti-TF antibody, and Abdesignates the anti-TF antibody. In one embodiment the linker-auristatinis vcMMAE.

In one embodiment the linker-conjugate is mcMMAF (where mc/MC is anabbreviation of maleimido caproyl)

wherein p denotes a number of from 1 to 8, e.g. p may be from 3-5, Srepresents a sulphydryl residue of the anti-TF antibody, and Abdesignates the anti-TF antibody.

In one embodiment the antibody blocks the binding of FVIIa to tissuefactor determined e.g. as described in Example 14.

In one embodiment the antibody inhibits FVIIa binding to tissue factor,preferably with a maximum value of inhibition IC₅₀ of between 0.01-3.0μg/mL, or such as 0.1-2.0 μg/mL, or such as 0.2-1.2 μg/mL whendetermined as described in Example 14.

In one embodiment the antibody competes for tissue factor binding

with an antibody comprising a VH region comprising the sequence of SEQID NO:33 and a VL region comprising the sequence of SEQ ID NO:73,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:1 and a VL region comprising the sequence of SEQ ID NO:41,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:5 and a VL region comprising the sequence of SEQ ID NO:45,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:9 and a VL region comprising the sequence of SEQ ID NO:49,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:13 and a VL region comprising the sequence of SEQ ID NO:53,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:17 and a VL region comprising the sequence of SEQ ID NO:57,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:21 and a VL region comprising the sequence of SEQ ID NO:61,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:25 and a VL region comprising the sequence of SEQ ID NO:65,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:29 and a VL region comprising the sequence of SEQ ID NO:69,

or

with an antibody comprising a VH region comprising the sequence of SEQID NO:37 and a VL region comprising the sequence of SEQ ID NO:77.

In one embodiment the antibody comprises:

-   -   a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:34, 35 and 36 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:74, 75 and 76, or    -   b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:2, 3 and 4 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:42, 43 and 44, or    -   c) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:6, 7 and 8 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:46, 47 and 48, or    -   d) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:10, 11 and 12 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:50, 51 and 52, or    -   e) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:14, 15 and 16 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:54, 55 and 56, or    -   f) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:18, 19 and 20 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:58, 59 and 60, or    -   g) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:22, 23 and 24 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:62, 63 and 64, or    -   h) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:26, 27 and 28 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:66, 67 and 68, or    -   i) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:30, 31 and 32 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:70, 71 and 72, or    -   j) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID        NO:38, 39 and 40 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO:78, 79 and 80, or    -   k) a variant of any of said antibodies, wherein said variant        preferably has at most 1, 2 or 3 amino-acid modifications, more        preferably amino-acid substitutions, such as conservative        amino-acid substitutions in said sequences.

In one embodiment the antibody comprises a VH having

-   -   a) at least 80% identity, such as at least 90%, at least 95%, or        at least 98% or 100% identity to a VH region sequence selected        from the group consisting of: SEQ ID NO:33, 1, 5, 9, 13, 17,        221, 25, 37 and 29, or    -   b) at most 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino-acid        modifications, more preferably amino-acid substitutions, such as        conservative amino-acid substitutions as compared to a VH region        sequence selected from the group consisting of: SEQ ID NO: 33,        1, 5, 9, 13, 17, 21, 25, 37 and 29.

In one embodiment the antibody comprises a VL having

-   -   a) at least 80% identity, such as at least 90%, at least 95%, or        at least 98% or 100% identity to a VL region sequence selected        from the group consisting of: SEQ ID NO:73, 41, 45, 49, 53, 57,        61, 65, 77 and 69, or    -   b) at most 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino-acid        modifications, more preferably amino-acid substitutions, such as        conservative amino-acid substitutions as compared to a VH region        sequence selected from the group consisting of: SEQ ID NO:73,        41, 45, 49, 53, 57, 61, 65, 77 and 69,

In one embodiment the antibody comprises:

-   -   a) a VH region comprising the sequence of SEQ ID NO:33 and a VL        region comprising the sequence of SEQ ID NO:73, or    -   b) a VH region comprising the sequence of SEQ ID NO:1 and a VL        region comprising the sequence of SEQ ID NO:41, or    -   c) a VH region comprising the sequence of SEQ ID NO:5 and a VL        region comprising the sequence of SEQ ID NO:45, or    -   d) a VH region comprising the sequence of SEQ ID NO:9 and a VL        region comprising the sequence of SEQ ID NO:49, or    -   e) a VH region comprising the sequence of SEQ ID NO:13 and a VL        region comprising the sequence of SEQ ID NO:53, or    -   f) a VH region comprising the sequence of SEQ ID NO:17 and a VL        region comprising the sequence of SEQ ID NO:57, or    -   g) a VH region comprising the sequence of SEQ ID NO:21 and a VL        region comprising the sequence of SEQ ID NO:61, or    -   h) a VH region comprising the sequence of SEQ ID NO:25 and a VL        region comprising the sequence of SEQ ID NO:65, or    -   i) a VH region comprising the sequence of SEQ ID NO:29 and a VL        region comprising the sequence of SEQ ID NO:69, or    -   j) a VH region comprising the sequence of SEQ ID NO:37 and a VL        region comprising the sequence of SEQ ID NO:77.

In one embodiment the antibody binds to the extracellular domain oftissue factor with an apparent affinity (EC₅₀) of 3.0 nM or less, suchas 0.50 nM or less, e.g. 0.35 nM or less, such as 0.20 nM or less, e.g.0.1 nM or less, when determined as described in the assay in Example 12.

In one embodiment the antibody binds to mammalian cells expressingtissue factor, such as A431 cells transfected with a construct encodingtissue factor, preferably with an apparent affinity (EC₅₀) of 10 nM orless, e.g. 8 nM or less, such as 5 nM or less, e.g. 2 nM or less, suchas 1 nM or less, e.g. 0.5 nM or less, such as 0.3 nM or less, whendetermined as described in the assay in Example 13.

In a another or alternative aspect the antibody is conjugated to atherapeutic moiety selected from the group consisting of taxol;cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin;etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin;daunorubicin; dihydroxy anthracin dione; a tubulin-inhibitor such asmaytansine or an analog or derivative thereof; mitoxantrone;mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocorticoid;procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicinor an analog or derivative thereof an antimetabolite such asmethotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, orcladribine; an alkylating agent such as mechlorethamine, thioepa,chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine(DTIC), procarbazine, mitomycin C, cisplatin, carboplatin, duocarmycinA, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivativethereof; pyrrolo[2,1-c][1,4] benzodiazepines (PDBs) or analoguesthereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin,doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,plicamycin, anthramycin (AMC)); diphtheria toxin and related moleculessuch as diphtheria A chain and active fragments thereof and hybridmolecules, ricin toxin such as ricin A or deglycosylated ricin A chaintoxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II SLT IIV,LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybeanBowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin,modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, Phytolacca americanaproteins such as PAPI, PAPII, and PAP S, momordica charantia inhibitor,curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase);DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein;diphtherin toxin; and Pseudomonas endotoxin. In a further alternativeembodiment the antibody, is conjugated to a cytotoxic moiety selectedfrom the group consisting of dolastatin, maytansine, calicheamicin,duocarmycin, rachelmycin (CC-1065), or an analog, derivative, or prodrugof any thereof.

In a further alternative embodiment the antibody is conjugated to acytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7,IL-10, IL-12, IL-13, IL-15, IL-18, TL-23, IL-27, IL-28a, IL-28b, IL-29,KGF, IFNα, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim,and TNFα.

In a further alternative embodiment the antibody is conjugated to aradioisotope.

In one embodiment the antibody is capable of inducing cytotoxicity byinternalization of the antibody coupled to a toxin in A431, BxPC3MDA-MB-23 as described in Example 15.

In one embodiment the antibody induces cytotoxicity by internalizationas described in Example 15, with an EC₅₀ value between 9×10⁻⁵ and 4×10⁻⁴μg/mL in A431 cells.

In one embodiment the preparation of the antibody drug conjugate resultsin less than 2%, such as less than 1.5%, or less than 1%, or less than0.5% unconjugated drug when determined as described in Example 16.

In one embodiment the preparation of the antibody drug conjugate resultsin less 10%, such as less than 8%, or less than 7% or less than 6% orless than 5.5% aggregates when determined as described in Example 16.

In one embodiment the preparation of the antibody drug conjugate resultsin less 1%, such as less than 0.5%, or less than 0.25%, or less than0.2% endotoxins when determined as described in Example 16.

In one embodiment the preparation of the antibody drug conjugate resultsin a concentration of antibody drug conjugate in the range of 1-100mg/mL, such as in the range of 2-50 mg/mL, or in the range of 5-25mg/mL, or in the range of 5-15 mg/mL, or in the range of 7.5-15 mg/mL,or in the range of 8-12 mg/mL, or in the range of 9-11 mg/mL whendetermined as described in Example 16.

In one embodiment the antibody drug conjugate binds to the extracellulardomain of tissue factor with an apparent affinity (EC₅₀) of 600 ng/mL orless, such as 550 ng/mL or less, or 500 ng/mL or less, such as with anEC₅₀ value in the range of 200-600 ng/mL, e.g. an EC₅₀ value in therange of 300-600 ng/mL, or an EC₅₀ value in the range of 350-550 ng/mL,or an EC₅₀ value in the range of 400-500 ng/mL, when determined asdescribed in Example 17.

In one embodiment the antibody drug conjugate induces cytotoxicity byinternalization as described in Example 18, with an EC₅₀ value between 1and 100 ng/mL in A431 cells.

In one embodiment the antibody drug conjugate induces cytotoxicity byinternalization as described in Example 18, with an EC₅₀ value between0.5 and 20 ng/mL in HPAF-II cells.

In one embodiment the antibody drug conjugate induces cytotoxicity byinternalization as described in Example 18, with an EC₅₀ value between0.5 and 500 ng/mL, such as between 0.5 and 20 ng/mL in NCI-H441 cells.

In one embodiment the antibody drug conjugate induces cytotoxicity byinternalization as described in Example 18, in e.g. tumor cells,expressing more than 200,000 tissue factor molecules per cell, such asbetween 200,000-1,000,000 tissue factor molecules per cell, e.g. between200,000 and 500,000 tissue factor molecules per cell. The EC₅₀ value mayin one embodiment be between 0.1 and 100 ng/mL.

In one embodiment the antibody drug conjugate induces cytotoxicity byinternalization as described in Example 18, in e.g. tumor cells,expressing more than 20,000 tissue factor molecules per cell, such asbetween 20,000-200,000 tissue factor molecules per cell. The EC₅₀ valuemay in one embodiment be between 0.5 and 500 ng/mL, such as between 0.5and 20 ng/mL.

In one embodiment the antibody drug conjugate inhibits tumour growth asdescribed in Example 19.

In one embodiment the antibody drug conjugate inhibits tumour growth ofa cell line expressing more than 1000 molecules tissue factor per cell,such as more than 10,000 tissue factor molecules per cells, e.g. morethan 100,000 tissue factor molecules per cell, or such as between1000-20,000 tissue factor molecules per cell, or between 20,000-200,000tissue factor molecules per cell, or between 200,000-500,000 tissuefactor molecules per cell, or between 200,000-1,000,000 tissue factormolecules per cell, when tumor growth is determined as described inExample 19.

In one embodiment the antibody drug conjugate is stable at −65° C. forat least three months, where stable refers to that at least 95% of theantibody drug conjugate is present as monomeric molecules whendetermined as described in Example 20.

In one embodiment the antibody drug conjugate is stable at 5° C. for atleast three months, where stable refers to that at least 95% of theantibody drug conjugate is present as monomeric molecules whendetermined as described in Example 20.

In another aspect the invention provides a pharmaceutical compositioncomprising the antibody drug conjugate as defined in any of the aboveembodiments. In one embodiment the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier.

In another aspect the invention provides the antibody drug conjugate asdefined in any of the above embodiments for use as a medicament.

In another aspect the invention provides the antibody drug conjugate asdefined in any of the above embodiments for use in the treatment of adisorder.

In another aspect the invention provides the antibody drug conjugate asdefined in any of the above embodiments for use in the treatment ofinflammation.

In another aspect the invention provides the antibody drug conjugate asdefined in any of the above embodiments for use in the treatment ofcancer.

In one embodiment, the cancer is selected from the group consisting oftumors of the central nervous system, head and neck cancer, lung cancer,such as NSCLC, breast cancer, specifically triple-negative breastcancer, esophageal cancer, gastric or stomach cancer, liver and biliarycancer, pancreatic cancer, colorectal cancer, bladder cancer, kidneycancer, prostate cancer, endometrial cancer, ovarian cancer, malignantmelanoma, sarcoma, tumors of unknown primary origin, bone marrow cancer,acute lymphoblastic leukemia, chronic lymphoblastic leukemia andnon-Hodgkin lymphoma, skin cancer, glioma, cancer of the brain, uterus,acute myeloid leukemia and rectum.

In one embodiment the cancer is pancreatic cancer.

In one embodiment the cancer is colorectal cancer.

In one embodiment the cancer is ovarian cancer.

In one embodiment the cancer is breast cancer.

In one embodiment the cancer is prostate cancer.

In one embodiment the cancer is bladder cancer.

In one embodiment, the cancer is a cancer which is sensitive totreatment with a tubulin inhibitor.

In another aspect the invention provides the antibody drug conjugate ofany one of above embodiments, wherein the medicament is for thetreatment of cancer in combination with one or more further therapeuticagents, such as a chemotherapeutic agent.

In another aspect the invention provides the use of the antibody drugconjugate of any one of the above embodiments for the manufacture of amedicament for the treatment of cancer. In one embodiment, the cancermay be selected from any one of the cancers described above.

In another aspect the invention provides a method for inducing celldeath, or inhibiting growth and/or proliferation of a tumor cellexpressing tissue factor, comprising administration, to an individual inneed thereof, of an effective amount of the antibody drug conjugate ofany of the above embodiments.

In another aspect the invention provides a method of treatment of any ofthe above cancer diseases by administration to an individual in needthereof, an effective amount of the antibody drug conjugate of any ofthe above embodiments. In one embodiment the antibody drug conjugate isadministered in combination with one or more further therapeutic agents,such as a chemotherapeutic agent,

Antibody

The present invention relates to anti-TF antibody drug conjugates, thuscomprising both an antibody and a drug, which may in particular beconjugated to each other via a linker.

The antibodies may be prepared by well known recombinant techniquesusing well known expression vector systems and host cells. In oneembodiment the antibodies are prepared in a CHO cell using the GSexpression vector system as disclosed in De la Cruz Edmunds et al.,2006, Molecular Biotechnology 34; 179-190, EP216846, U.S. Pat. No.5,981,216, WO 87/04462, EP323997, U.S. Pat. No. 5,591,639, U.S. Pat. No.5,658,759, EP338841, U.S. Pat. No. 5,879,936, and U.S. Pat. No.5,891,693.

After isolating and purifying the antibodies from the cell media usingwell known techniques they are conjugated with the auristatin via alinker as further disclosed below.

Monoclonal antibodies of the present invention may e.g. be produced bythe hybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature 352,624-628 (1991) and Marks et al., 3. Mol, Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against tissue factor may begenerated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. Such transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene minilocus thatencodes unrearranged human heavy (μ and γ) and K light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar.D., Intern. Rev. Immunol, Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann, N.Y. Acad. Sci 764 536-546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International Immunology5, 647-656 (1993), Tuaillon at al., J. Immunol, 152, 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,877,397,U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S. Pat. No.5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,545,807, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al, EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo12 human heavy chain transgene (as described in Example 2 of WO01/1.4424).

The HCo17 transgenic mouse strain (see also US 2010/0077497) wasgenerated by coinjection of the 80 kb insert of pHC2 (Taylor et al.(1994) int. Immunol., 6: 579-591), the Kb insert of pVX6, and a −460 kbyeast artificial chromosome fragment of the yIgH24 chromosome. This linewas designated (HCo17) 25950. The (HCo17) 25950 line was then bred withmice comprising the CMD mutation (described in Example 1 of PCTPublication WO 01109187), the JKD mutation (Chen et al, (1993) EMBO J12: 811-820), and the (KC05) 9272 transgene (Fishwild et al. (1996)Nature Biotechnology 14: 845-851). The resulting mice express humanimmunoglobulin heavy and kappa light chain trans genes in a backgroundhomozygous for disruption of the endogenous mouse heavy and kappa lightchain loci.

The HCo20 transgenic mouse strain is the result of a co-injection ofminilocus 30 heavy chain transgene pHC2, the germline variable region(Vh)-containing YAC yIgH10, and the minilocus construct pVx6 (describedin WO09097006). The (HCo20) line was then bred with mice comprising theCMD mutation (described in Example 1 of PCT Publication WO 01/09187),the JKD mutation (Chen et al. (1993) EMBO J. 12: 811-820), and the(KCO5) 9272 trans gene (Fishwild eta). (1996) Nature Biotechnology 14:845-851). The resulting mice express human 10 immunoglobulin heavy andkappa light chain transgenes in a background homozygous for disruptionof the endogenous mouse heavy and kappa light chain loci.

In order to generate HuMab mice with the salutary effects of the Balb/cstrain, HuMab mice were crossed with KCO05 [MIK] (Balb) mice which weregenerated by backcrossing the KC05 strain (as described in Fishwild et(1996) Nature Biotechnology 14:845-851) to wild-type Balb/c mice togenerate mice as described in WO09097006. Using this crossing Balb/chybrids were created for HCo12, HCo17, and HCo20 strains.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187, Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according to wellknown techniques, Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. No. 5,827,690, U.S. Pat. No.5,756,687, U.S. Pat. No. 5,750,172 and U.S. Pat. No. 5,741,957.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be generated throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, and other techniques, usingtechniques well known in the art and the resulting molecules may besubjected to additional maturation, such as affinity maturation, as suchtechniques are well known in the art (see for instance Hoogenboom etal., 3. Mol, Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-4085 (1993), Hogenboom et al., Immunol, Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant regions, kappa or lambda, maybe used. If desired, the class of an anti-TF antibody of the presentinvention may be switched by known methods. For example, an antibody ofthe present invention that was originally IgM may be class switched toan IgG antibody of the present invention. Further, class switchingtechniques may be used to convert one IgG subclass to another, forinstance from IgG1 to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1,κ.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody.The term full-length antibody is intended to be understood as referringto what is generally known as a natural, whole, antibody, i.e. not afragment or other types of antibodies where the different chains of anantibody has been re-arranged by man to generate a new type of antibody(see e.g. Sidhu S S, Nature Biotechnology, 25, 5, 537-538, (2007)disclosing full-length antibodies on display). In another embodiment,the antibody of the invention is an antibody fragment or a single-chainantibody.

Antibody fragments may e.g. be obtained by fragmentation usingconventional techniques, and the fragments screened for utility in thesame manner as described herein for whole antibodies. For example,F(ab′)₂ fragments may be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment may be treated to reduce disulfide bridges toproduce Fab′ fragments, Fab fragments may be obtained by treating an IgGantibody with papain; Fab′ fragments may be obtained with pepsindigestion of IgG antibody. A F(ab′) fragment may also be produced bybinding Fab′ described below via a thioether bond or a disulfide bond. AFab′ fragment is an antibody fragment obtained by cutting a disulfidebond of the hinge region of the F(ab′)₂. A Fab′ fragment may be obtainedby treating a F(ab′)₂ fragment with a reducing agent, such asdithiothreitol. Antibody fragments may also be generated by expressionof nucleic acids encoding such fragments in recombinant cells (see forinstance Evans et al., J. Immunol, Meth. 184, 123-38 (1995)). Forexample, a chimeric gene encoding a portion of a F(ab′)₂ fragment couldinclude DNA sequences encoding the C_(H)1 domain and hinge region of theH chain, followed by a translational stop codon to yield such atruncated antibody fragment molecule.

The antibodies of the present invention are further disclosed andcharacterized in WO 2010/066803 (Genmab A/S).

In one embodiment the anti-TF antibody is a stabilized IgG4 antibody,Examples of suitable stabilized IgG4 antibodies are antibodies, whereinarginine at position 409 in a heavy chain constant region of human IgG4,which is indicated in the EU index as in Kabat et al., is substitutedwith lysine, threonine, methionine, or leucine, preferably lysine(described in WO2006033386 (Kirin)) and/or wherein the hinge regioncomprises a Cys-Pro-Pro-Cys sequence.

In a further embodiment, the stabilized IgG4 anti-TF antibody is an IgG4antibody comprising a heavy chain and a light chain, wherein said heavychain comprises a human IgG4 constant region having a residue selectedfrom the group consisting of: Lys, Ala, Thr, Met and Leu at the positioncorresponding to 409 and/or a residue selected from the group consistingof: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, andwherein said antibody optionally comprises one or more furthersubstitutions, deletions and/or insertions, but does not comprise aCys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibodycomprises a Lys or Ala residue at the position corresponding to 409 orthe CH3 region of the antibody has been replaced by the CH3 region ofhuman IgG1, of human IgG2 or of human IgG3.

In an even further embodiment, the stabilized IgG4 anti-TF antibody isan IgG4 antibody comprising a heavy chain and a light chain, whereinsaid heavy chain comprises a human IgG4 constant region having a residueselected from the group consisting of: Lys, Ala, Thr, Met and Leu at theposition corresponding to 409 and/or a residue selected from the groupconsisting of: Ala, Val, Gly, Ile and Leu at the position correspondingto 405, and wherein said antibody optionally comprises one or morefurther substitutions, deletions and/or insertions and wherein saidantibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region.Preferably, said antibody comprises a Lys or Ala residue at the positioncorresponding to 409 or the CH3 region of the antibody has been replacedby the CH3 region of human IgG1, of human IgG2 or of human IgG3.

In a further embodiment, the anti-TF antibody is an antibody of anon-IgG4 type, e.g. IgG1, IgG2 or IgG3 which has been mutated such thatthe ability to mediate effector functions, such as ADCC, has beenreduced or even eliminated. Such mutations have e.g. been described inDall'Acqua W F et al., J. Immunol. 177(2):1129-1138 (2006) and HezarehM, J Virol.; 75(24):12161-12168 (2001),

Conjugates

The present invention provides an anti-TF antibody drug conjugate.

In one aspect the anti-TF antibody drug conjugates of the presentinvention comprise an anti-TF antibody as disclosed herein conjugated toauristatins or auristatin peptide analogs and derivates (U.S. Pat. No.5,635,483; U.S. Pat. No. 5,780,588). Auristatins have been shown tointerfere with microtubule dynamics, GTP hydrolysis and nuclear andcellular division (Woyke et al (2001) Antimicrob, Agents and Chemother.45(12): 3580-3584) and have anti-cancer (U.S. Pat. No. 5,663,149) andanti-fungal activity (Pettit et al, (1998) Antimicrob. Agents andChemother, 42:2961-2965, The auristatin drug moiety may be attached tothe antibody via a linker, through the N (amino) terminus or the C(terminus) of the peptidic, drug moiety.

Exemplary auristatin embodiments include the N-terminus-linkedmonomethyl auristatin drug moieties DE and DF, disclosed in Senter etal., Proceedings of the American Association for Cancer Research, Volume45, abstract number 623, presented Mar. 28, 2004 and described in US2005/0238649).

An exemplary auristatin embodiment is MMAE (monomethyl auristatin E),wherein the wavy line indicates the covalent attachment to the linker(L) of an antibody drug conjugate:

Another exemplary auristatin embodiment is MMAF (monomethyl auristatinF), wherein the wavy line indicates the covalent attachment to a linker(L) of an antibody drug conjugate (US2005/0238649):

The anti-TF antibody drug conjugates according to the invention comprisea linker unit between the cytostatic or cytotoxic drug unit and theantibody unit. In some embodiments, the linker is cleavable underintracellular conditions, such that the cleavage of the linker releasesthe drug unit from the antibody in the intracellular environment. In yetanother embodiment, the linker unit is not cleavable and the drug is forinstance released by antibody degradation. In some embodiments, thelinker is cleavable by a cleavable agent that is present in theintracellular environment (e.g. within a lysosome or endosome orcaveola). The linker can be, e.g. a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including but not limitedto, a lysosomal or endosomal protease. In some embodiments, the peptidyllinker is at least two amino acids long or at least three amino acidslong. Cleaving agents can include cathepsins B and D and plasmin, all ofwhich are known to hydrolyze dipeptide drug derivatives resulting in therelease of active drug inside the target cells (see e.g. Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment,the peptidyl linker cleavable by an intracellular protease is a Val-Cit(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker(see e.g. U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Val-Cit linker and different examples of Phe-Lyslinkers). Examples of the structures of a Val-Cit and a Phe-Lys linkerinclude but are not limited to MC-vc-PAB described below, MC-vc-GABA,MC-Phe-Lys-PAB or MC-Phe-Lys-GABA, wherein MC is an abbreviation formaleimido caproyl, vc is an abbreviation for Val-Cit, PAB is anabbreviation for p-aminobenzylcarbamate and GABA is an abbreviation forγ-aminobutyric acid. An advantage of using intracellular proteolyticrelease of the therapeutic agent is that the agent is typicallyattenuated when conjugated and the serum stabilities of the conjugatesare typically high.

In yet another embodiment, the linker unit is not cleavable and the drugis released by antibody degradation (see US 2005/0238649). Typically,such a linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment” in the context of a linker means that no morethan 20%, typically no more than about 15%, more typically no more thanabout 10%, and even more typically no more than about 5%, no more thanabout 3%, or no more than about 1% of the linkers, in a sample ofantibody drug conjugate compound, are cleaved when the antibody drugconjugate compound presents in an extracellular environment (e.g.plasma). Whether a linker is not substantially sensitive to theextracellular environment can be determined for example by incubatingthe antibody drug conjugate compound with plasma for a predeterminedtime period (e.g. 2, 4, 8, 16 or 24 hours) and then quantitating theamount of free drug present in the plasma.

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components have the following structures (wherein Ab meansantibody and p, representing the drug-loading (or average number ofcytostatic or cytotoxic drugs per antibody molecule), is 1 to about 8,e.g. p may be from 4-6, such as from 3-5, or p may be 1, 2, 3, 4, 5, 6,7 or 8).

Examples where a cleavable linker is combined with an auristatin includeMC-vc-PAB-MMAF (also designated as vcMMAF) and MC-vc-PAB-MMAF (alsodesignated as vcMMAE), wherein MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for the Val-Cit (valine-citruline) basedlinker, and PAB is an abbreviation for p-aminobenzylcarbamate:

Other examples include auristatins combined with a non-cleavable linker,such as mcMMAF (mc (MC is the same as mc in this context) is anabbreviation of maleimido caproyl):

The cytostatic or cytotoxic drug loading is represented by p and is theaverage number of cytostatic drug moieties per antibody in a molecule(also designated as the drug to antibody ratio, DAR). The cytostatic orcytotoxic drug loading may range from 1 to 20 drug moieties per antibodyand may occur on amino acids with useful functional groups such as, butnot limited to, amino or sulfhydryl groups, as in lysine or cysteine.

Depending on the way of conjugation, p may be limited by the number ofattachment sites on the antibody, for example where the attachment is asulphydryl group, as in the present invention. Generally, antibodies donot contain many sulphydryl groups, (free and reactive cysteine thiolgroups) which may be linked to a drug moiety as most cysteine thiolresidues in antibodies exist as disulfide bridges. Therefore, in certainembodiments, an antibody may be reduced with reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor fully reducing conditions, to generate reactive sulphydryl residues,in certain embodiments, the drug loading for an ADC of the inventionranges from 1 to about 8, e.g. p may be from 4-6, such as from 3-5, or pmay be 1, 2, 3, 4, 5, 6, 7, or 8, as a maximum of 8 sulphydryl residuesbecomes available after (partial) reduction of the antibody (there are 8cysteines involved in inter-chain disulfide bonding).

In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE druglinker moiety and conjugation methods are disclosed in WO2004010957,U.S. Pat. No. 7,659,241, U.S. Pat. No. 7,829,531, U.S. Pat. No.7,851,437 and U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (whichare incorporated herein by reference), and the vcMMAE drug linker moietyis bound to the anti-TF antibodies at the cysteines using a methodsimilar to those disclosed in therein.

In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF druglinker moiety and conjugation methods are disclosed in U.S. Pat. No.7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711 (Seattle Genetics,Inc.), (which are incorporated herein by reference), and the mcMMAF druglinker moiety is bound to the anti-TF antibodies at the cysteines usinga method similar to those disclosed in therein.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-011-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-098-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-111-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-114-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-011-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-098-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-111-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate isHuMab-TF-114-mcMMAF.

In an alternative embodiment the anti-TF antibody is conjugated to atherapeutic moeity, such as a cytotoxin, a chemotherapeutic drug, acytokine, an immunosuppressant, or a radioisotope. Such conjugates arereferred to herein as “immunoconjugates”. Immunoconjugates which includeone or more cytotoxins are referred to as “immuno-toxins”.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to(e.g., kills) cells. Suitable therapeutic agents for formingimmunoconjugates of the present invention include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, maytansine or an analog orderivative thereof, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin, calicheamicin or analogs or derivativesthereof; antimetabolites (such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents(such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin; as well asduocarmycin A, duocarmycin SA, CC-1065 (a.k.a. rachelmycin), or analogsor derivatives of CC-1065), dolastatin, pyrrolo[2,1-c][1,4]benzodiazepins (PDBs) or analogues thereof, antibiotics (such asdactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerlydaunomycin), doxorubicin, idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g.,tubulin-inhibitors), such as diphtheria toxin and related molecules(such as diphtheria A chain and active fragments thereof and hybridmolecules); ricin toxin (such as ricin A or a deglycosylated ricin Achain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-TI,SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanustoxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin,alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaccaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, and enomycin toxins. Othersuitable conjugated molecules include antimicrobialilytic peptides suchas CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease (RNase),DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan atal., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal forClinicians 44, 43 (1994). Therapeutic agents that may be administered incombination with anti-TF antibody drug conjugates of the presentinvention as described elsewhere herein, such as, e.g., anti-cancercytokines or chemokines, are also candidates for therapeutic moietiesuseful for conjugation to an antibody disclosed in the presentinvention.

In another alternative embodiment, an anti-TF antibody drug conjugatedisclosed in the present invention comprises a conjugated nucleic acidor nucleic acid-associated molecule. In one such embodiment, theconjugated nucleic acid is a cytotoxic ribonuclease, an antisensenucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or animmunostimulatory nucleic acid (e.g., an immunostimulatory CpGmotif-containing DNA molecule). In another alternative embodiment, aanti-TF antibody of the invention is conjugated to an aptamer or aribozyme instead of an auristatin or a functional peptide analog orderivate thereof.

In another alternative embodiment, anti-TF antibody drug conjugatescomprising one or more radiolabeled amino acids are provided. Aradiolabeled anti-TF antibody may be used for both diagnostic andtherapeutic purposes (conjugation to radiolabeled molecules is anotherpossible feature). Non-limiting examples of labels for polypeptidesinclude 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methodsfor preparing radiolabeled amino acids and related peptide derivativesare known in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581, U.S. Pat.No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S.RE:35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902. Forexample, a radioisotope may be conjugated by a chloramine T method.

In one embodiment, the antibody is conjugated to a radioisotope or to aradioisotope-containing chelate. For example, the antibody can beconjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, whichallows for the antibody to be complexed with a radioisotope. Theantibody may also or alternatively comprise or be conjugated to one ormore radiolabeled amino acids or other radiolabeled molecule. Aradiolabeled anti-TF antibody may be used for both diagnostic andtherapeutic purposes. Non-limiting examples of radioisotopes include ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹²⁵I, ¹¹¹In, ¹³¹I, ¹⁸⁶Re, ²¹³ Bs, ²²⁵AC and²²²Th.

Anti-TF antibodies may also be chemically modified by covalentconjugation to a polymer to for instance increase their circulatinghalf-life. Exemplary polymers, and methods to attach them to peptides,are illustrated in for instance U.S. Pat. No. 4,766,106, U.S. Pat. No.4,179,337, U.S. Pat. No. 4,495,285 and U.S. Pat. No. 4,609,546,Additional polymers include polyoxyethylated polyols and polyethyleneglycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000and about 40,000, such as between about 2,000 and about 20,000). Thismay for example be used if the anti-TF antibody is a fragment.

Any method known in the art for conjugating the anti-TF antibodydisclosed in the present invention to the conjugated molecule(s), suchas those described above, may be employed, including the methodsdescribed by Hunter et al., Nature 144, 945 (1962), David et al.,Biochemistry 13, 1014 (1974), Pain et Immunol. Meth. 40, 219 (1981) andNygren, 3, Histochem. and Cytochem, 30, 407 (1982). Such antibodies maybe produced by chemically conjugating the other moiety to the N-terminalside or C-terminal side of the anti-TF antibody or fragment thereof(e.g., a anti-TF antibody H or L chain) (see, e.g., Antibody EngineeringHandbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)).Such conjugated antibody derivatives may also be generated byconjugation at internal residues or sugars, where appropriate.

The agents may be coupled either directly or indirectly to a anti-TFantibody disclosed in the present invention. One example of indirectcoupling of a second agent is coupling via a spacer moiety to cysteineor lysine residues in the antibody. In one embodiment, an anti-TFantibody is conjugated, via a spacer or linker, to a prodrug moleculethat can be activated in vivo to a therapeutic drug. Afteradministration, the spacers or linkers are cleaved by tumorcell-associated enzymes or other tumor-specific conditions, by which theactive drug is formed. Examples of such pro-drug technologies andlinkers are described in WO02083180, WO2004043493, WO2007018431,WO2007089149, WO2009017394 and WO201062171 by Syntarga By, et al, (allincorporated herein by reference) Suitable antibody-pro-drug technologyand duocarmycin analogs can also be found in U.S. Pat. No. 6,989,452(Medarex) (incorporated herein by reference).

In one embodiment, the anti-TF antibody of the present invention isattached to a chelator linker, e.g. tiuxetan, which allows for theantibody to be conjugated to a radioisotope.

In a further aspect, the invention relates to an expression vectorencoding an antibody of the invention. For example if the anti-TFantibody of the present invention is conjugated to a therapeutic moietydifferent than an auristatin or a functional peptide analog or derivatethereof. Such expression vectors may in one embodiment be used toexpress the anti-TF antibody of the present invention which may thensubsequently be conjugated to a moiety as described herein.

In one embodiment, the expression vector of the invention comprises anucleotide sequence encoding one or more of the amino acid sequencesselected from the group consisting of: SEQ ID NO:1-4, 5-8, 33-36, 37-40,41-44, 45-48, 73-76 and 77-80.

In another particular embodiment, the expression vector of the inventioncomprises a nucleotide sequence encoding one or more of the VH aminoacid sequences selected from the group consisting of: SEQ ID NO: 1, 5,37 and 33.

In a particular embodiment, the expression vector of the inventioncomprises a nucleotide sequence encoding one or more of the VH CDR3amino acid sequences selected from the group consisting of: SEQ ID NO 4,8, 40 and 36.

In another particular embodiment, the expression vector of the inventioncomprises a nucleotide sequence encoding one or more of the VL aminoacid sequences selected from the group consisting of; SEQ ID NO: 41, 45,77 and 73.

In another embodiment, the expression vector of the invention comprisesa nucleotide sequence encoding one or more of the VL CDR3 amino acidsequences selected from the group consisting of: SEQ ID NO: 44, 48, 80and 76.

In a particular embodiment the expression vector of the inventioncomprises a nucleotide sequence encoding variants of one or more of theabove amino acid sequences, said variants having at most 25 amino acidmodifications, such 20, such as at most 15, 14, 13, 12 or 11 amino acidmodifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino-acidmodifications, such as deletions or insertions, preferablysubstitutions, such as conservative substitutions or at least 80%identity to any of said sequences, such as at least 85% identity or 90%identity or 95% identity, such as 96% identity or 97% identity or 98%identity or 99% identity to any of the afore mentioned amino acidsequences.

In a further embodiment, the expression vector further comprises anucleotide sequence encoding the constant region of a light chain, aheavy chain or both light and heavy chains of an antibody, e.g. a humanantibody.

Such expression vectors may be used for recombinant production ofantibodies of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-TF antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in for instance Sykes and Johnston, Nat Biotech 17, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in for instance Schakowski et al., Mol Ther 3,793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaPO4-precipitated construct (as described in for instance WO00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler etal., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics7, 603 (1981)). Such nucleic acid vectors and the usage thereof are wellknown in the art (see for instance U.S. Pat. No. 5,589,466 and U.S. Pat.No. 5,973,972).

In one embodiment, the vector is suitable for expression of the anti-TFantibody in a bacterial cell. Examples of such vectors includeexpression vectors such as BlueScript (Stratagene), pIN vectors (VanHeeke & Schuster, Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen,Madison Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed,Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516-544 (1987)).

A nucleic acid and/or vector may also comprises a nucleic acid sequenceencoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the art,and include secretion leader or signal peptides, organelle targetingsequences (e.g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e.g., stop transfer sequences,GPI anchor sequences), and the like.

In an expression vector of the invention, anti-TF antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g.,polylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In one embodiment, the anti-TF-antibody-encoding expression vector maybe positioned in and/or delivered to the host cell or host animal via aviral vector.

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an anti-TF antibody of the invention as defined herein or abispecific molecule of the invention as defined herein. Examples of hostcells include yeast, bacterial and mammalian cells, such as CH0 or HEKcells. For example, in one embodiment, the present invention provides acell comprising a nucleic acid stably integrated into the cellulargenome that comprises a sequence coding for expression of an anti-TFantibody of the present invention. In another embodiment, the presentinvention provides a cell comprising a non-integrated nucleic acid, suchas a plasmid, cosmid, phagemid, or linear expression element, whichcomprises a sequence coding for expression of an anti-TF antibody of theinvention.

In a further aspect, the invention relates to a hybridoma which producesan antibody of the invention as defined herein. In an even furtheraspect, the invention relates to a transgenic non-human animalcomprising nucleic acids encoding a human heavy chain and a human lightchain, wherein the animal or plant produces an antibody of the inventionof the invention. Generation of such hybridomas and transgenic animalshas been described above.

In a further aspect, the invention relates to a method for producing ananti-TF antibody of the invention, said method comprising the steps of

a) culturing a hybridoma or a host cell of the invention as describedherein above, and

b) purifying the antibody of the invention from the culture media andoptionally

c) transforming the anti-TF antibody into an ADC.

Pharmaceutical Composition

Upon purifying the anti-TF antibody drug conjugates they may beformulated into pharmaceutical compositions using well knownpharmaceutical carriers or excipients.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the antibodydrug conjugate of the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition of the present invention(e.g., less than a substantial impact (10% or less relative inhibition,5% or less relative inhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

Cancer cells overexpressing TF may be particularly good targets for theanti-TF antibody drug conjugates of the invention, since more antibodiesmay be bound per cell. Thus, in one embodiment, a cancer patient to betreated with an anti-TF antibody drug conjugate of the invention is apatient, e.g. a pancreatic cancer, lung cancer or colorectal cancerpatient who has been diagnosed to have one or more mutations in K-rasand/or one or more mutations in p53 in their tumor cells. TF expressionis under control of two major transforming events driving diseaseprogression (activation of K-ras oncogene and inactivation of the p53tumor suppressor), in a manner dependent on MEK/mitogen-activatedprotein kinase (MAPK) and phosphatidylinositol 3′-kinase (PI3K) (Yu etal. (2005) Blood 105:1734).

The actual dosage levels of the antibody drug conjugate in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the antibody drug conjugate which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the amide thereof,the route of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular compositions employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering an antibody drug conjugate ofthe present invention are well known in the art and may be selected bythose of ordinary skill in the art.

In one embodiment, the pharmaceutical composition of the presentinvention is administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment the pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with antibody drugconjugate of the present invention.

Examples of suitable aqueous- and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe anti-TF antibody drug conjugate of the present invention, usethereof in the pharmaceutical compositions of the present invention iscontemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions of the present invention may alsocomprise pharmaceutically acceptable antioxidants for instance (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of the present invention may alsocomprise isotonicity agents, such as sugars, polyalcohols, such asmannitol, sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Theanti-TF antibody drug conjugate of the present invention may be preparedwith carriers that will protect the compound against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Such carriers mayinclude gelatin, glyceryl monostearate, glyceryl distearate,biodegradable, biocompatible polymers such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid alone or with a wax, or other materials well known inthe art. Methods for the preparation of such formulations are generallyknown to those skilled in the art. See e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978.

In one embodiment, the anti-TF antibody drug conjugate of the presentinvention may be formulated to ensure proper distribution in vivo.Pharmaceutically acceptable carriers for parenteral administrationinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or nonaqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the anti-TF antibody drug conjugate in the required amountin an appropriate solvent with one or a combination of ingredients e.g.as enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe anti-TF antibody drug conjugate into a sterile vehicle that containsa basic dispersion medium and the required other ingredients e.g. fromthose enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, examples of methods ofpreparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating theanti-TF antibody drug conjugate in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the anti-TF antibody drugconjugate into a sterile vehicle that contains a basic dispersion mediumand the required other ingredients from those enumerated above. In thecase of sterile powders for the preparation of sterile injectablesolutions, examples of methods of preparation are vacuum drying andfreeze-drying (lyophilization) that yield a powder of the anti-TFantibody drug conjugate plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The pharmaceutical composition of the present invention may contain oneanti-TF antibody drug conjugate of the present invention or acombination of anti-TF antibody drug conjugates of the presentinvention.

As described above, in another aspect, the invention relates to theanti-TF antibody drug conjugate of the invention as defined herein foruse as a medicament.

The anti-TF antibody drug conjugates of the invention may be used for anumber of purposes. In particular, the anti-TF antibody drug conjugatesof the invention may be used for the treatment of various forms ofcancer, in one aspect the anti-TF antibody drug conjugates of theinvention are used for the treatment of various solid cancer types suchas: tumors of the central nervous system, head and neck cancer, lungcancer (such as non-small cell lung cancer), breast cancer cancer (suchas triple-negative breast cancer), esophageal cancer, stomach cancer,liver and biliary cancer, pancreatic cancer, colorectal cancer, bladdercancer, kidney cancer, prostate cancer, endometrial cancer, ovariancancer, malignant melanoma, sarcoma (soft tissue eg, bone and muscle),tumors of unknown primary origin (i.e. unknown primaries), leukemia,bone marrow cancer (such as multiple myeloma) acute lymphoblasticleukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma, acutemyeloid leukemia (AML), skin cancer, glioma, cancer of the brain,uterus, and rectum.

Further autoimmune inflammation, such as myopathies or multiplesclerosis may be targeted with the anti-TF antibody drug conjugates ofthe present invention.

Cancer related hemostatic disorders may also be targeted with thepresent invention.

Further diseases with inflammation, such as myopathies, rheumatoidarthritis, osteoarthritis, ankylosing spondylitis, gout,spondylarthropathris, ankylosing spondylitis, Reiter's syndrome,psoriatic arthropathy, enterapathric spondylitis, juvenile arthropathy,reactive arthropathy, infectious or post-infectious arthritis,tuberculous arthritis, viral arthritis, fungal arthritis, syphiliticarthritis, glomerulonephritis, end stage renal disease, systemic lupuserythematosus, mb. Crohn, ulcerative colitis, inflammatory boweldisease, cystic fibrosis, chronic obstructive pulmonary disease (COPD),astma, allergic astma, bronchitis, acute bronchiolitis, chronicbronchiolitis, idiopathic pulmonary fibrosis, or multiple sclerose maybe targeted with the anti-TF antibodies of the present invention.

Also vascular diseases such as vascular restenosis, myocardial vasculardisease, cerebral vascular disease, retinopathia and maculardegeneration, including but not limited to wet AMD can be treated withanti-TF antibody drug conjugates.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for the treatment of patients with cardiovascular risk, suchas atherosclerosis, hypertension, diabetis, dyslipiclemia, and acutecoronary syndrome, including but not limited to Acute MyocardialInfarct, stroke.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for inhibition of thrombosis, such as DVT, renal embolism,lung embolism, arterial thrombosis, or to treat thrombosis occurringfollowing arteriel surgical, peripheral vascular bypass grafts orcoronary artery bypass grafts, arterio-venous shunts, removal of animplementation, such as a stent or catheter.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for inhibition of renal ischemic reperfusion injury.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for treatment of hyperlipoproteineimia or hyperparathyroidism.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for treatment of vasculitis, ANCA-positive vasculitis orBehcet's disease.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for blocking traume-induced respiratory failure, such as acuterespiratory distress syndrome or acute lung injury.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for blocking infection-induced organ dysfunction, such asrenal failure, acute respiratory distress syndrome, or acute lunginjury.

The anti-TF antibody drug conjugates of the present invention may alsobe useful to treat various thromboembolic disorders such as thosearising from angioplasty, myocardial infarction, unstable angina andcoronary artery stenoses.

The anti-TF antibody drug conjugates of the present invention may alsobe useful in a prophylactic setting to treat TF-mediated complicationsto systemic infections, such as sepsis or pneumonia.

The anti-TF antibody drug conjugates of the present invention may alsobe useful as prophylactic treatment of patients with atheroscleroticvessels at risk for thrombosis.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for treatment of graft-versus-host disease.

The anti-TF antibody drug conjugates of the present invention may alsobe useful for increasing beta cell engraftment in islet transplantation,to prevent cardiac allograft vasculopathy (CAV) and to prevent acutegraft rejection.

Similarly, the invention relates to a method for inhibiting growthand/or proliferation of a tumor cell expressing TF, comprisingadministration, to an individual in need thereof, of an anti-TF antibodydrug conjugate of the invention. In one embodiment, said tumor cell isinvolved in cancer, such as prostate cancer, lung cancer (such asnon-small cell lung cancer), breast cancer (such as triple-negativebreast cancer), colorectal cancer (such as metastatic colorectalcancer), pancreatic cancer, endometrial cancer, ovarian cancer,cutaneous melanoma, leukemia bone marrow cancer (such as multiplemyeloma), acute lymphoblastic leukemia, chronic lymphoblastic leukemiaand non-Hodgkin lymphoma, skin cancer, prostate cancer, glioma, cancerof the brain, kidneys, uterus, bladder, acute myeloid leukemia (AML) andrectum.

Also, the invention relates to the use of anti-TF antibody drugconjugates that bind to human TF for the preparation of a medicament forthe treatment of cancer, such as one of the specific cancer indicationsmentioned above.

In an embodiment selection of patients to be treated with anti-TFantibody drug conjugate is based on the level of TF in their urineand/or blood. In a particular embodiment the patient to be treated has arelatively high level of TF in urine and/or blood. For example, thepatient to be treated may have a TF level in urine of more than 20ng/mL, such as more than 40 ng/mL, e.g. more than 100 mg/mL, such asmore than 200 ng/mL, Alternatively, or in addition, the TF level inserum of the patients may be more than 100 pg/mL, such as more than 200pg/mL. This may e.g. be determined using an ELISA. Methods for doingthis include but are not limited to those described below in relation todiagnostic uses.

However, it is also within the scope of the present invention to treatpatients with anti-TF antibody drug conjugate of the present inventionwhich has a lower level of TF in the urine and/or blood.

In one embodiment selection of patients to be treated with anti-TFantibody drug conjugates of the present invention may be based on thelevel of TF expression. The level of TF expression may be evaluated byexposing the patients to a radiolabeled anti-TF antibody and thenmeasuring the level of radioactivity in the patients. The radiolabeledanti-TF antibody may be an anti-TF antibody described in the presentinvention, i.e. an antibody of the anti-TF antibody drug conjugatesdescribed herein, or it may be another anti-TF antibody. Examples ofradiolabels may be any of those described above in relation toradiolabeling of antibodies. Methods for doing this include but are notlimited to those described below in relation to diagnostic uses.

In a further embodiment of the methods of treatment of the presentinvention, the efficacy of the treatment is being monitored during thetherapy, e.g. at predefined points in time. In one embodiment, theefficacy may be monitored by measuring the level of TF in urine orblood, for example by ELISA. Methods for doing this include but are notlimited to those described below in relation to diagnostic uses. Inanother embodiment, the efficacy may be determined by visualization ofthe disease area, e.g. by performing one or more PET-CT scans, forexample using a labeled anti-TF antibody, such as a labeled anti-TFantibody described in the present invention. Furthermore, labeledanti-TF antibodies, such as labeled anti-TF antibodies 011, 098, 114 and111 disclosed herein, could be used to detect TF-producing tumors e.g.using a PET-CT scan.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired therapeutic response. For example, asingle dose may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subjects to be treated; each unit contains a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the anti-TF antibodydrug conjugates depend on the disease or condition to be treated and maybe determined by the persons skilled in the art. An exemplary,non-limiting range for a therapeutically effective amount of a compoundof the present invention is about 0.1-100 mg/kg, such as about 0.1-50mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, suchas about 0.5-5 mg/kg, for instance about 5 mg/kg, such as about 4 mg/kg,or about 3 mg/kg, or about 2 mg/kg, or about 1 mg/kg, or about 0.5mg/kg, or about 0.3 mg/kg. An exemplary, non-limiting range for atherapeutically effective amount of an anti-TF antibody drug conjugateof the present invention is about 0.02-30 mg/kg, such as about 0.1-20mg/kg, or about 0.5-10 mg/kg, or about 0.5-5 mg/kg, for example about1-2 mg/kg, in particular of the antibodies 011, 098, 114 or 111 asdisclosed herein.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of the anti-TFantibody drug conjugate employed in the pharmaceutical composition atlevels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, a suitable daily dose of a compositionof the present invention will be that amount of the compound which isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous, and for instance administered proximal to the site ofthe target. If desired, the effective daily dose of a pharmaceuticalcomposition may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. While it is possible for ananti-TF antibody drug conjugate of the present invention to beadministered alone, it is preferable to administer the anti-TF antibodydrug conjugate as a pharmaceutical composition as described above.

In one embodiment, the anti-TF-antibody drug conjugate may beadministered by infusion in a weekly dosage of from 10 to 1500 mg/m²,such as from 30 to 1500 mg/m², or such as from 50 to 1000 mg/m², or suchas from 10 to 500 mg/m², or such as of from 100 to 300 mg/m². Suchadministration may be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the anti-TF antibody drug conjugates may beadministered by infusion every third week in a dosage of from 30 to 1500mg/m², such as of from 50 to 1000 mg/m² or 100 to 300 mg/m². Suchadministration may be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the anti-TF antibody drug conjugates may beadministered by slow continuous infusion over a long period, such asmore than 24 hours, in order to reduce toxic side effects.

In one embodiment the anti-TF antibody drug conjugates may beadministered in a weekly dosage of 50 mg to 2000 mg, such as for example50 mg, 100 mg, 200 mg, 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000mg, for up to 16 times, such as from 4 to 10 times, such as from 4 to 6times. The administration may be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of anti-TF antibody drug conjugate of the presentinvention in the blood upon administration, by for instance taking out abiological sample and using anti-idiotypic antibodies which target theantigen binding region of the anti-TF antibody drug conjugates of thepresent invention.

In one embodiment, the anti-TF antibody drug conjugate may beadministered by maintenance therapy, such as, e.g., once a week for aperiod of 6 months or more.

In one embodiment, the anti-TF antibody drug conjugates may beadministered by a regimen including one infusion of an anti-TF antibodydrug conjugate of the present invention followed by an infusion of ananti-TF antibody of the present invention, such as antibody 011, 098,114 or 111 disclosed herein containing a radioisotope. The regimen maybe repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a weekly, bi-weekly, three-weekly or monthly dosageof a anti-TF antibody drug conjugate of the present invention in anamount of about 0.1-100 mg/kg, such as 0.3-3 mg/kg, e.g. 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60,70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, oralternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or insome cases week 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

An “effective amount” for tumor therapy may also be measured by itsability to stabilize the progression of disease. The ability of acompound to inhibit cancer may be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition may be evaluated by examining the ability of the compoundto inhibit cell growth or to induce apoptosis by in vitro assays knownto the skilled practitioner. A therapeutically effective amount of atherapeutic compound may decrease tumor size, or otherwise amelioratesymptoms in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

An anti-TF antibody drug conjugate may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission. This may beespecially useful in patients wherein it is difficult to locate a tumorthat is known to be present due to other biological factors.

The anti-TF antibody drug conjugate may also be administered incombination therapy, i.e., combined with other therapeutic agentsrelevant for the disease or condition to be treated. Accordingly, in oneembodiment, the anti-TF antibody drug conjugate medicament is forcombination with one or more further therapeutic agents, such as acytotoxic, chemotherapeutic or anti-angiogenic agent.

Such combined administration may be simultaneous, separate orsequential. For simultaneous administration, the agents may beadministered as one composition or as separate compositions, asappropriate. The present invention thus also provides methods fortreating a disorder involving cells expressing TF as described above,which methods comprise administration of an anti-TF antibody drugconjugate of the present invention combined with one or more additionaltherapeutic agents as described below.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate of the present invention and at leastone chemotherapeutic agent to a subject in need thereof.

In one embodiment, the present invention provides a method for treatingor preventing cancer, which method comprises administration of atherapeutically effective amount of an anti-TF antibody drug conjugateof the present invention and at least one chemotherapeutic agent to asubject in need thereof.

In one embodiment, the present invention provides the use of an anti-TFantibody drug conjugate of the present invention for the preparation ofa pharmaceutical composition to be administered with at least onechemotherapeutic agent for treating cancer.

In one embodiment, such a chemotherapeutic agent may be selected from anantimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea,asparaginase, gemcitabine, cladribine and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from analkylating agent, such as mechlorethamine, thioepa, chlorambucil,melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide,busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC),procarbazine, mitomycin C, cisplatin and other platinum derivatives,such as carboplatin, and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from ananti-mitotic agent, such as taxanes, for instance docetaxel, andpaclitaxel, and vinca alkaloids, for instance vindesine, vincristine,vinblastine, and vinorelbine.

In one embodiment, such a chemotherapeutic agent may be selected from atopoisomerase inhibitor, such as topotecan or irinotecan.

In one embodiment, such a chemotherapeutic agent may be selected from acytostatic drug, such as etoposide and teniposide.

In one embodiment, such a chemotherapeutic agent may be selected from agrowth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such asIressa, erbitux (cetuximab), tarceva and similar agents), an inhibitorof ErbB2 (Her2/neu) (such as herceptin and similar agents) and similaragents.

In one embodiment, such a chemotherapeutic agent may be selected from atyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571),lapatinib, PTK787/ZK222584 and similar agents.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate of the present invention and at leastone inhibitor of angiogenesis, neovascularization, and/or othervascularization to a subject in need thereof.

Examples of such angiogenesis inhibitors are urokinase inhibitors,matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY12-9566, AG 3340, BMS-275291 and similar agents), inhibitors ofendothelial cell migration and proliferation (such as TNP-470,squalamine, 2-methoxyestradiol, combretastatins, endostatin,angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison,N.J.), R115777 (Janssen Pharmaceutical, Inc, Titusville, N.J.) andsimilar agents), antagonists of angiogenic growth factors (such asZD6474, SU6668, antibodies against angiogenic agents and/or theirreceptors (such as VEGF, bFGF, and angiopoietin-1), thalidomide,thalidomide analogs (such as CC-5013), Sugen 5416, SU5402,antiangiogenic ribozyme (such as angiozyme), interferon α (such asinterferon α2a), suramin and similar agents), VEGF-R kinase inhibitorsand other anti-angiogenic tyrosine kinase inhibitors (such as SU011248),inhibitors of endothelial-specific integrin/survival signaling (such asvitaxin and similar agents), copper antagonists/chelators (such astetrathiomolybdate, captopril and similar agents), carboxyamido-triazole(CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as wellas nucleotide molecules inhibiting angiogenesis (such asantisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53and cDNA coding for deficient VEGF receptor-2) and similar agents.

Other examples of such inhibitors of angiogenesis, neovascularization,and/or other vascularization are anti-angiogenic heparin derivatives andrelated molecules (e.g., heparinase III), temozolomicle, NK4, macrophagemigration inhibitory factor (MIF), cyclooxygenase-2 inhibitors,inhibitors of hypoxia-inducible factor 1, anti-angiogenic soyisoflavones, oltipraz, fumagillin and analogs thereof, somatostatinanalogues, pentosan polysulfate, tecogalan sodium, dalteparin,tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin,antibodies against other relevant targets (such as anti-alpha-v/beta-3integrin and anti-kininostatin mAbs) and similar agents.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate of the present invention for treatingthe disorders as described above may be an anti-cancer immunogen, suchas a cancer antigen/tumor-associated antigen (e.g., epithelial celladhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonicantigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100,Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines(e.g., human papillomavirus vaccines), tumor-derived heat shockproteins, and similar agents. A number of other suitable cancerantigens/tumor-associated antigens described elsewhere herein andsimilar molecules known in the art may also or alternatively be used insuch embodiment. Anti-cancer immunogenic peptides also includeanti-idiotypic “vaccines” such as BEC2 anti-idiotypic antibodies,Mitumomab, CeaVac and related anti-idiotypic antibodies, anti-idiotypicantibody to MG7 antibody, and other anti-cancer anti-idiotypicantibodies (see for instance Birebent et al., Vaccine, 21(15), 1601-12(2003), Li et al., Chin Pled j (Engl). 114(9), 962-6 (2001), Schmitt etal., Hybridoma. 13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3),191-209 (1985), Raychardhuri et al., 3 Immunol. 137(5), 1743-9 (1986),Pohl et al., Int Cancer. 50(6), 958-67 (1992), Bohlen et al., CytokinesMol Them. 2(4), 231-8 (1996) and Maruyama, J Immunol Methods, 264(1-2),121-33 (2002)). Such anti-idiotypic Abs may optionally be conjugated toa carrier, which may be a synthetic (typically inert) molecule carrier,a protein (for instance keyhole limpet hemocyanin (KLH) (see forinstance Ochi et al., Eur Immunol, 17(11), 1645-8 (1987)), or a cell(for instance a red blood cell—see for instance Wi et al., J ImmunolMethods. 122(2), 227-34 (1989)).

In one embodiment of the invention, the anti-TF antibody drug conjugateis combined with an immuno-oncology drug such as Yervoy (ipilimumab)which potentially acts by inducing T cell immunity against the cancer,Cytoreduction with the anti-TF antibody drug conjugate in combinationwith an immunostimulatory drug might provide significant clinicalbenefit to patients.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate of the present invention for treatingthe disorders as described above may be an anti-cancer cytokine,chemokine, or combination thereof. Examples of suitable cytokines andgrowth factors include TFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12,IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF,IFNα (e.g., INFα2b). TFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor,ancestim, and TNFα. Suitable chemokines may include Glu-Leu-Arg(ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from thehuman CXC and C—C chemokine families. Suitable cytokines includecytokine derivatives, cytokine variants, cytokine fragments, andcytokine fusion proteins. These and other methods or uses involvingnaturally occurring peptide-encoding nucleic acids herein mayalternatively or additionally be performed by “gene activation” andhomologous recombination gene upregulation techniques, such as aredescribed in U.S. Pat. No. 5,968,502, U.S. Pat. No. 6,063,630 and U.S.Pat. No. 6,187,305 and EP 0505500.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be a cell cyclecontrol/apoptosis regulator (or “regulating agent”). A cell cyclecontrol/apoptosis regulator may include molecules that target andmodulate cell cycle control/apoptosis regulators such as (i) cdc-25(such as NSC 663284), (ii) cyclin-dependent kinases that overstimulatethe cell cycle (such as flavopiriciol (L868275, HMR1275),7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine(R-roscovitine, CYC202)), and (iii) telomerase modulators (such asBIBR1532, SOT-095, GRN163 and compositions described in for instanceU.S. Pat. No. 6,440,735 and U.S. Pat. No. 6,713,055), Non-limitingexamples of molecules that interfere with apoptotic pathways includeTNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand(Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-senseBcl-2.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be a hormonal regulatingagent, such as agents useful for anti-androgen and anti-estrogentherapy. Examples of such hormonal regulating agents are tamoxifen,idoxifene, fulvestrant, droloxifene, toremifene, raloxifene,diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such asflutaminde/eulexin), a progestin (such as such as hydroxyprogesteronecaproate, medroxyprogesterone/provera, rnegestrol acepate/megace), anadrenocorticosteroid (such as hydrocortisone, prednisone), luteinizinghormone-releasing hormone (and analogs thereof and other LHRH agonistssuch as buserelin and goserelin), an aromatase inhibitor (such asanastrazole/arimidex, aminoglutethimide/cytraden, exemestane), a hormoneinhibitor (such as octreotide/-sandostatin) and similar agents.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be an anti-anergic agent(for instance small molecule compounds, proteins, glycoproteins, orantibodies that break tolerance to tumor and cancer antigens). Examplesof such compounds are molecules that block the activity of CTLA-4, suchas MDX-010/Yervoy (ipilimumab) (Phan et al., PNAS USA 100, 8372 (2003)),which potentially acts by inducing T cell immunity against the cancer.Cytoreduction with the anti-TF antibody drug conjugate in combinationwith an immunostimulatory drug might provide significant clinicalbenefit to patients.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be a tumor suppressorgene-containing nucleic acid or vector such as a replication-deficientadenovirus encoding human recombinant wild-type p53/SCH58500, etc.;antisense nucleic acids targeted to oncogenes, mutated, or deregulatedgenes; or siRNA targeted to mutated or deregulated genes. Examples oftumor suppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4(Smad4), MSH2, MLH1, and DCC.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be an anti-cancer nucleicacid, such as genasense (augrnerosen/G3139), LY900003 (ISIS 3521), ISIS2503, OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome encapsulatedc-raf antisense oligonucleotide/ISIS-5132), MG98, and other antisensenucleic acids that target PKCα, clusterin, IGEBPs, protein kinase A,cyclin D1, or Bcl-2h.

In one embodiment, a therapeutic agent for use in combination with ananti-TF antibody drug conjugate according to the present invention fortreating the disorders as described above may be an anti-cancerinhibitory RNA molecule (see for instance Lin et al., Curr Cancer DrugTargets. 1(3), 241-7 (2001), Erratum in: Curr Cancer Drug Targets. 3(3),237 (2003), Lima et al., Cancer Gene Ther, 11(5), 309-16 (2004), Grzmilet al., Int J Oncol, 4(1), 97-105 (2004), Collis et al., Int RadiatOncol Biol Phys. 57(2 Suppl), S144 (2003), Yang et al., Oncogene.22(36), 5694-701 (2003) and Zhang et al., Biochem Biophys Res Commun.303(4), 1169-78 (2003)).

Compositions and combination administration methods of the presentinvention also include the administration of nucleic acid vaccines, suchas naked DNA vaccines encoding such cancer antigens/tumor-associatedantigens (see for instance U.S. Pat. No. 5,589,466, U.S. Pat. No.5,593,972, U.S. Pat. No. 5,703,057, U.S. Pat. No. 5,879,687, U.S. Pat.No. 6,235,523, and U.S. Pat. No. 6,387,888). In one embodiment, thecombination administration method and/or combination compositioncomprises an autologous vaccine composition. In one embodiment, thecombination composition and/or combination administration methodcomprises a whole cell vaccine or cytokine-expressing cell (for instancea recombinant IL-2 expressing fibroblast, recombinantcytokine-expressing dendritic cell, and the like) (see for instanceKowalczyk et al., Acta Biochim Pol. 5)(3), 613-24 (2003), Reilly et al.,Methods Mol. Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther,2(1), 79-89 (2002). Another example of such an autologous cell approachthat may be useful in combination methods of the present invention isthe MyVax® Personalized Immunotherapy method (previously referred to asGTOP-99) (Genitope Corporation—Redwood City, Calif., USA).

In one embodiment, the present invention provides combinationcompositions and combination administration methods wherein an anti-TFantibody drug conjugate according to the present invention is combinedor co-administered with a virus, viral proteins, and the like.Replication-deficient viruses, that generally are capable of one or onlya few rounds of replication in vivo, and that are targeted to tumorcells, may for instance be useful components of such compositions andmethods. Such viral agents may comprise or be associated with nucleicacids encoding immunostimulants, such as GM-CSF and/or IL-2. Bothnaturally oncolytic and such recombinant oncolytic viruses (for instanceHSV-1 viruses, reoviruses, replication-deficient andreplication-sensitive adenovirus, etc.) may be useful components of suchmethods and compositions. Accordingly, in one embodiment, the presentinvention provides combination compositions and combinationadministration methods wherein an anti-TF antibody drug conjugate iscombined or co-administered with an oncolytic virus. Examples of suchviruses include oncolytic adenoviruses and herpes viruses, which may ormay not be modified viruses (see for instance Shah et al. Neurooncol.65(3), 203-26 (2003), Stiles et al., Surgery. 134(2), 357-64 (2003),Sunarmura et al., Pancreas. 28(3), 326-9 (2004), Teshigahara et al.,Sorg Oncol. 85(1), 42-7 (2004), Varghese et al., Cancer Gene Ther.9(12), 967-78 (2002), Wildner et al., Cancer Res. 59(2), 410-3 (1999),Yamanaka, Int 3 Oncol. 24(4), 919-23 (2004) and Zwiebel et al., SeminOncol. 28(4), 336-43 (2001).

Combination compositions and combination administration methods of thepresent invention may also involve “whole cell” and “adoptive”immunotherapy methods. For instance, such methods may comprise infusionor re-infusion of immune system cells (for instance tumor-infiltratinglymphocytes (TILs), such as CD4⁺ and/or CD8⁺ cells (for instance T cellsexpanded with tumor-specific antigens and/or genetic enhancements),antibody-expressing B cells or other antibody producing/presentingcells, dendritic cells (DCs) (e.g., anti-cytokine expressing recombinantdendritic cells, dendritic cells cultured with a DC-expanding agent suchas GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loadeddendritic cells), anti-tumor NK cells, so-called hybrid cells, orcombinations thereof. Cell lysates may also be useful in such methodsand compositions. Cellular “vaccines” in clinical trials that may beuseful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96(Antigenics), and Melacine® cell lysates. Antigens shed from cancercells, and mixtures thereof (see for instance Bystryn et al., ClinicalCancer Research Vol. 7, 1882-1887, July 2001), optionally admixed withadjuvants such as alum, may also be components in such methods andcombination compositions.

In one embodiment, an anti-TF antibody drug conjugate according to thepresent invention may be delivered to a patient in combination with theapplication of an internal vaccination method. Internal vaccinationrefers to induced tumor or cancer cell death, such as drug-induced orradiation-induced cell death of tumor cells, in a patient, thattypically leads to elicitation of an immune response directed towards(i) the tumor cells as a whole or (ii) parts of the tumor cellsincluding (a) secreted proteins, glycoproteins or other products, (b)membrane-associated proteins or glycoproteins or other componentsassociated with or inserted in membranes, and/or (c) intracellularproteins or other intracellular components. An internalvaccination-induced immune response may be humoral (i.e.antibody-complement-mediated) or cell-mediated (e.g., the developmentand/or increase of endogenous cytotoxic T lymphocytes that recognize theinternally killed tumor cells or parts thereof). In addition toradiotherapy, non-limiting examples of drugs and agents that may be usedto induce said tumor cell-death and internal vaccination areconventional chemotherapeutic agents, cell-cycle inhibitors,anti-angiogenesis drugs, monoclonal antibodies, apoptosis-inducingagents, and signal transduction inhibitors.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with an anti-TF antibody drugconjugate according to the present invention for treating the disordersas described above are differentiation inducing agents, retinoic acidanalogues (such as all trans retinoic acid, 13-cis retinoic acid andsimilar agents), vitamin D analogues (such as seocalcitol and similaragents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa,PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met,Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with the anti-TF antibody drugconjugate according to the present invention for treating the disordersas described above are cathepsin B, modulators of cathepsin Ddehydrogenase activity, glutathione-S-transferase (such asglutacylcysteine synthetase and lactate dehydrogenase), and similaragents.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with an anti-TF antibody drugconjugate according to the present invention for treating the disordersas described above are estramustine and epirubicin.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with an anti-TF antibody drugconjugate according to the present invention for treating the disordersas described above are a HSP90 inhibitor like 17-allyl aminogeld-anamycin, antibodies directed against a tumor antigen such as PSA,CA125, KSA, etc., integrins like integrin β1, inhibitors of VCAM andsimilar agents.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with an anti-TF antibody drugconjugate according to the present invention for treating the disordersas described above are caicineurin-inhibitors (such as valspodar, PSC833 and other MDR-1 or p-glycoprotein inhibitors), TOR-inhibitors (suchas sirolimus, everolimus and rapamcyin), and inhibitors of “lymphocytehoming” mechanisms (such as FTY720), and agents with effects on cellsignaling such as adhesion molecule inhibitors (for instance anti-LEA,etc.).

In one embodiment, the anti-TF antibody drug conjugate of the inventionis for use in combination with one or more other therapeutic antibodies,such as bevacizumab (Avastin®), zalutumumab, cetuximab (Erbitux®),paniturnurnab (Vectibix™), ofatumumab (Arzerra®), zanolimumab,daratumumab (HuMax-CD38), ranibizumab (Lucentis®), daclizumab(Zenapax®), basiliximab (Simulect®), infliximab (Remicad®), adalimumab(Humira®), natalizumab (Tysabri®), omalizumab (Xolair®), efalizumab(Raptiva®), nimotuzumab, rituxirnab (Rituxan®/MabThera®) and/ortrastuzumab (Herceptin®). Other therapeutic antibodies which may be usedin combination with the anti-TF antibody drug conjugate of the presentinvention are those disclosed in WO98/40408 (antibodies that can bindnative human TF), WO04/094475 (antibodies capable of binding to humantissue factor, which do not inhibit factor mediated blood coagulationcompared to a normal plasma control), WO03/093422 (antibodies that bindwith greater affinity to the TF:VIIa complex than to TF alone),WO03/037361 (TF agonist or antagonist for treatment related toapoptosis) or WO 2010/066803 (human monoclonal antibodies against tissuefactor).

In one embodiment, the anti-TF antibody drug conjugate may beadministered in connection with the delivery of one or more agents thatpromote access of the anti-TF antibody drug conjugate or combinationcomposition to the interior of a tumor. Such methods may for example beperformed in association with the delivery of a relaxin, which iscapable of relaxing a tumor (see for instance U.S. Pat. No. 6,719,977).In one embodiment, an anti-TF antibody drug conjugate of the presentinvention may be bonded to a cell penetrating peptide (CPP). Cellpenetrating peptides and related peptides (such as engineered cellpenetrating antibodies) are described in for instance Zhao et al.,Immunol Methods. 254(1-2), 137-45 (2001), Hong et al., Cancer Res,60(23), 6551-6 (2000). Lindgren et al., Biochem 377(Pt 1), 69-76 (2004),Buerger et al., J Cancer Res Clin Oncol, 129(12), 669-75 (2003), Poogaet al., FASEB 1, 12(1), 67-77 (1998) and Tseng et al., Mol. Pharmacol.62(4), 864-72 (2002).

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate of the present invention and at leastone anti-inflammatory agent to a subject in need thereof.

In one embodiment such an anti-inflammatory agent may be selected fromaspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib andcelecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac,diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac,oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies(e.g. antibodies described in WO2004058797, e.g. 10F8), anti-IL15antibodies (e.g. antibodies described in WO03017935 and WO2004076620),anti-IL15R antibodies, anti-CD4 antibodies (e.g. zanolimumab),anti-CD11a antibodies (e.g., efalizumab), anti-alpha-4/beta-1 integrin(VLA4) antibodies (e.g. natalizumab), CTLA4-Ig for the treatment ofinflammatory diseases, prednisolone, prednisone, disease modifyingantirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine,sulfasalazine, pyrimidine synthesis inhibitors (such as leflunomide),IL-1 receptor blocking agents (such as anakinra), TNF-α blocking agents(such as etanercept, infliximab, and adalimumab) and similar agents.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate of the present invention and at leastone immunosuppressive and/or immunomodulatory agent to a subject in needthereof.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from cyclosporine, azathioprine, mycophenolicacid, mycophenolate mofetil, corticosteroids such as prednisone,methotrexate, gold salts, sulfasalazine, antimalarials, brequinar,leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine,cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocytethymopentin, thymosin-o and similar agents.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from immunosuppressive antibodies, such asantibodies binding to p75 of the IL-2 receptor, antibodies against CD25(e.g. those described in WO2004045512, such as A81, AB7, AB11, andAB12), or antibodies binding to for instance MHC, CD2, CD3, CD4, CD7,CD28, 87, CD40, CD45, IFNγR, INFαR or TNFR (consists of two subunits:CD120a and CD120b), IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, CD11a, orCD58, or antibodies binding to theft ligands.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from soluble IL-15R, IL-10R, B7 molecules (B7-1,67-2, variants thereof, and fragments thereof), ICOS, and OX40, aninhibitor of a negative T cell regulator (such as an antibody againstCTLA4) and similar agents.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate according to the present invention andan anti-C3b(i) antibody to a subject in need thereof.

In one embodiment, a therapeutic agent for use in combination with theanti-TF antibody drug conjugates for treating the disorders as describedabove may be selected from histone deacetylase inhibitors (for instancephenylbutyrate) and/or DNA repair agents (for instance DNA repairenzymes and related compositions such as dimericine).

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-011-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-098-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-111-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-114-vcMMAE.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-011-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-098-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-111-mcMMAF.

In one embodiment, the anti-TF antibody drug conjugate for use incombination therapy with any one of the above mentioned agents isHuMab-TF-114-mcMMAF.

Methods of the present invention for treating a disorder as describedabove comprising administration of a therapeutically effective amount ofan anti-TF antibody drug conjugate according to the present inventionmay also comprise anti-cancer directed photodynamic therapy (forinstance anti-cancer laser therapy—which optionally may be practicedwith the use of photosensitizing agent, see, for instance Zhang et al.,3 Control Release. 93(2), 141-50 (2003)), anti-cancer sound-wave andshock-wave therapies (see for instance Kambe et al., Hum Cell. 10(1),87-94 (1997)), and/or anti-cancer nutraceutical therapy (see forinstance Roudebush et al., Vet Clin North Am Small Anim Pract, 34(1),249-69, viii (2004) and Rafi, Nutrition, 20(1), 78-82 (2004). Likewise,an anti-TF antibody drug conjugate may be used for the preparation of apharmaceutical composition for treating a disorder as described above tobe administered with anti-cancer directed photodynamic therapy (forinstance anti-cancer laser therapy—which optionally may be practicedwith the use of photosensitizing agent, anti-cancer sound-wave andshock-wave therapies, and/or anti-cancer nutraceutical therapy, in oneembodiment, the present invention provides a method for treating adisorder involving cells expressing TF in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-TF antibody drug conjugate, such as an anti-TF antibody drugconjugate of the present invention, and radiotherapy to a subject inneed thereof.

In one embodiment, the present invention provides a method for treatingor preventing cancer, which method comprises administration of atherapeutically effective amount of an anti-TF antibody drug conjugateof the present invention, and radiotherapy to a subject in need thereof.

In one embodiment, the present invention provides the use of an anti-TFantibody drug conjugate, of the present invention, for the preparationof a pharmaceutical composition for treating cancer to be administeredin combination with radiotherapy.

Radiotherapy may comprise radiation or administration ofradiopharmaceuticals to a patient. The source of radiation may be eitherexternal or internal to the patient being treated (radiation treatmentmay, for example, be in the form of external beam radiation therapy(EBRT) or brachytherapy (BT)). Radioactive elements that may be used inpracticing such methods include, e.g., radium, cesium-137, iridium-192,americium-241, gold-198, cobalt-57, copper-67, technetium-99,iodide-123, iodide-131, and indium-111.

In a further embodiment, the present invention provides a method fortreating or preventing cancer, which method comprises administration toa subject in need thereof of a therapeutically effective amount of ananti-TF antibody drug conjugate of the present invention, in combinationwith surgery.

As described above, a pharmaceutical composition of the presentinvention may be administered in combination therapy, i.e., combinedwith one or more agents relevant for the disease or condition to betreated either as separate pharmaceutical compositions or with acompound of the present invention co-formulated with one or moreadditional therapeutic agents as described above. Such combinationtherapies may require lower dosages of the compound of the presentinvention and/or the co-administered agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

In addition to the above, other relevant combination therapies includethe following:

-   -   For the treatment of pancreatic cancer an anti-TF antibody drug        conjugate according to the present invention in combination with        an antimetabolite, such as 5-fluorouracil and/or gemcitabine,        possibly in combination with one or more compounds selected        from: 90Y-hPAM4, ARC-100, ARQ-197, AZO-6244, bardoxolone methyl,        cixutumumab, (IMC-A12), folitixorin calcium, GVAX, ipilimumab,        KRX-0601, merbarone, MGCD-0103, MORAb-009, PX-12, Rh-Apo2L,        TLN-4601, trabedersen, volociximab (M200), WX-674, pemetrexed,        rubitecan, ixabepilone, OCX-0191Vion, 216586-46-8, lapatinib,        matuzumab, imatinib, sorafinib, trastuzurnab, exabepilone,        erlotinib, avastin and cetuximab    -   For the treatment of colorectal cancer an anti-TF antibody drug        conjugate according to the present invention in combination with        one or more compounds selected from: gemcitabine, bevacizumab,        FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, irinotecan, 5-FU/LV,        Capecitabine, UFT, EGFR-targeting agents, such as cetuximab,        panitumumab, nimotuzumab, zalutumumab; VEGF inhibitors, or        tyrosine kinase inhibitors such as sunitinib.    -   For the treatment of breast cancer an anti-TF antibody drug        conjugate according to the present invention in combination with        one or more compounds selected from: antimetabolites,        anthracyclines, taxanes, alkylating agents, epothilones        anti-hormonal (femar, tamoxifen etc), inhibitors of ErbB2        (Her2/neu) (such as herceptin and similar agents), CAF/FAC        (cyclofosfamide, doxorubicine, 5FU) AC (cyclo, doxo), CMF        (cyclo, methotrexate, 5FU), Docetaxel f capecitabine, GT        (paclitaxel, gemcitabine) FEC (cyclo, epi, 5FU) in combination        with herceptine: Paclitaxel +/− carboplatin, Vinorelbine,        Docetaxel, Conn. in combination with lapatinib; Capecitabine.    -   For the treatment of bladder an anti-TF antibody drug conjugate        according to the present invention in combination with one or        more compounds selected from: antimetabolites (gemcitabine,        alimta, methotrexate), platinum analogues (cisplatin,        carboplatin), EGFr inhibitors (such as cetuximab or        zalutumumab), VEGF inhibitors (such as Avastin) doxorubicin,        tyrosine kinase inhibitors such as gefitinib, trastuzumab,        anti-mitotic agent, such as taxanes, for instance paclitaxel,        and vinca alkaloids, for instance vinblastine.    -   For the treatment of prostate cancer an anti-TF antibody drug        conjugate according to the present invention in combination with        one or more compounds selected from: hormonal/antihormonal        therapies; such as antiandrogens, luteinizing hormone releasing        hormone (LHRH) agonists, and chemotherapeutics such as taxanes,        mitoxantrone, estramustine, 5FU, vinblastine, ixabepilone.    -   For the treatment of ovarian cancer an anti-TF antibody drug        conjugate according to the present invention in combination with        one or more compounds selected from; an anti-mitotic agent, such        as taxanes, and vinca alkaloids, caelyx, topotecan.        Diagnostic Uses

The anti-TF antibodies of the invention may also be used for diagnosticpurposes. The anti-TF antibodies described herein may in one embodimentbe conjugated to a detection agent or label instead of a drug, therebymaking them suitable for diagnostic purpose. In one embodiment thediagnostic use of an anti-TF antibody or anti-TF antibody conjugated toa detection agent may be used in combination with one of the othermethods of the present invention, in particular a pharmaceutical use ofthe anti-TF antibody drug conjugate of the present invention. Anti-TFantibody conjugated to a detection agent may in some cases allow for adirect detection of binding of the anti-TF antibody to TF, examples of“detection agent” or “label” are given in the following and reference to“anti-TF antibody” in the following may where relevant also includereference to “anti-TF antibody conjugated to a detection agent orlabel”. The term “diagnostic uses” includes also measuring the level ofTF in e.g. plasma, urine or expression levels of TF in biopsies inrelation to selecting patients for treatment or measuring the efficacyof a treatment as described above, and the use of e.g. radiolabelledanti-TF antibodies for e.g. selecting patients for treatment asdescribed above. Thus, in a further aspect, the invention relates to adiagnostic composition comprising an anti-TF antibody as defined herein,wherein the diagnostic composition may in a particular embodiment beused in combination with an anti-TF antibody drug conjugate of thepresent invention.

In one embodiment, the anti-TF antibodies of the present invention maybe used in vivo or in vitro for diagnosing diseases wherein cellsexpressing TF play an active role in the pathogenesis, by detectinglevels of TF, or levels of cells which contain TF on their membranesurface. This may be achieved, for example, by contacting a sample to betested, optionally along with a control sample, with the anti-TFantibody under conditions that allow for formation of a complex betweenthe anti-TF antibody and TF. Complex formation is then detected (e.g.,using an ELISA). When using a control sample along with the test sample,complex is detected in both samples and any statistically significantdifference in the formation of complexes between the samples isindicative of the presence of TF in the test sample.

Thus, in a further aspect, the anti-TF antibodies of the presentinvention may also be used in a method for detecting the presence of TFantigen, or a cell expressing TF, in a sample comprising:

contacting the sample with an anti-TF antibody of the invention or abispecific molecule of the invention, under conditions that allow forformation of a complex between the antibody and TF; and

analyzing whether a complex has been formed.

In one embodiment, the method is performed in vitro.

More specifically, the anti-TF antibodies of present invention may alsobe used in methods for the identification of, and diagnosis of invasivecells and tissues, and other cells targeted by anti-TF antibodies of thepresent invention, and for the monitoring of the progress of therapeutictreatments, status after treatment, risk of developing cancer, cancerprogression, and the like.

In one example of such a diagnostic assay, the anti-TF antibodies ofpresent invention may be used in a method of diagnosing the level ofinvasive cells in a tissue. Such a method comprises forming animmunocomplex between an anti-TF antibody and potential TF-containingtissues, and detecting formation of the immunocomplex, wherein theformation of the immunocomplex correlates with the presence of invasivecells in the tissue. The contacting may be performed in vivo, usinglabeled isolated antibodies and standard imaging techniques, or may beperformed in vitro on tissue samples.

The anti-TF antibodies of the present invention may also be used todetect TF-containing peptides and peptide fragments in any suitablebiological sample by any suitable technique. Examples of conventionalimmunoassays provided by the present invention include, withoutlimitation, an ELISA, an RIA, FACS assays, plasmon resonance assays,chromatographic assays, tissue immunohistochemistry, Western blot,and/or immunoprecipitation using an anti-TF antibody, Anti-TF antibodiesof the present invention may be used to detect TF and TF-fragments fromhumans. Suitable labels for the anti-TF antibody and/or secondaryantibodies used in such techniques include, without limitation, variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, p-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S, and ³H.

Anti-TF antibodies may also be used for assaying in a biological sampleby a competition immunoassay utilizing TF peptide standards labeled witha detectable substance and an unlabeled anti-TF antibody. In such anassay, the biological sample, the labeled TF peptide standard(s) and theanti-TF antibodies are combined and the amount of labeled TF standardbound to the unlabeled anti-TF antibody is determined. The amount of TFpeptide in the biological sample is inversely proportional to the amountof labeled TF standard bound to the anti-TF antibody.

The anti-TF antibodies are particularly useful in the in vivo imaging oftumors. In vivo imaging of tumors associated with TF may be performed byany suitable technique. For example, ⁹⁹Tc-labeling or labeling withanother gamma-ray emitting isotope may be used to label anti-TFantibodies in tumors or secondary labeled (e.g., FITC labeled) anti-TFantibody:TF complexes from tumors and imaged with a gamma scintillationcamera (e.g., an Elscint Apex 409ECT device), typically usinglow-energy, high resolution collimator or a low-energy all-purposecollimator. Stained tissues may then be assessed for radioactivitycounting as an indicator of the amount of TF-associated peptides in thetumor. The images obtained by the use of such techniques may be used toassess biodistribution of TF in a patient, mammal, or tissue, forexample in the context of using TF or TF-fragments as a biomarker forthe presence of invasive cancer cells. Variations on this technique mayinclude the use of magnetic resonance imaging (MRI) to improve imagingover gamma camera techniques. Similar immunoscintigraphy methods andprinciples are described in, e.g., Srivastava (ed.), RadiolabeledMonoclonal Antibodies For Imaging And Therapy (Plenum Press 1988),Chase, “Medical Applications of Radioisotopes,” in Remington'sPharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp.624-652 (Mack Publishing Co., 1990), and Brown, “Clinical Use ofMonoclonal Antibodies,” in Biotechnology And Pharmacy 227-49, Pezzuto etal., (eds.) (Chapman & Hall 1993). Such images may also be used fortargeted delivery of other anti-cancer agents, examples of which aredescribed herein (e.g., apoptotic agents, toxins, or CHOP chemotherapycompositions). Moreover, such images may also or alternatively serve asthe basis for surgical techniques to remove tumors. Furthermore, such invivo imaging techniques may allow for the identification andlocalization of a tumor in a situation where a patient is identified ashaving a tumor (due to the presence of other biomarkers, metastases,etc.), but the tumor cannot be identified by traditional analyticaltechniques. All of these methods are features of the present inventionand such methods may in particular be used in combination with treatmentof a patient with an anti-TF antibody drug conjugate of the presentinvention.

The in vivo imaging and other diagnostic methods provided by the presentinvention are particularly useful in the detection of micrometastases ina human patient (e.g., a patient not previously diagnosed with cancer ora patient in a period of recovery/remission from a cancer). Carcinomacancer cells, which may make up to 90% of all cancer cells, for example,have been demonstrated to stain very well with anti-TF antibodycompositions. Detection with monoclonal anti-TF antibodies describedherein may be indicative of the presence of carcinomas that areaggressive/invasive and also or alternatively provide an indication ofthe feasibility of using related monoclonal anti-TF antibody againstsuch micrometastases.

In one embodiment, the anti-TF antibodies of the present invention maybe used in an in vivo imaging method wherein an anti-TF antibody of thepresent invention is conjugated to a detection-promoting radio-opaqueagent, the conjugated antibody is administered to a host, such as byinjection into the bloodstream, and the presence and location of thelabeled antibody in the host is assayed. Through this technique and anyother diagnostic method provided herein, the anti-TF antibodies of thepresent invention May be used in a method for screening for the presenceof disease-related cells in a human patient or a biological sample takenfrom a human patient.

For diagnostic imaging, radioisotopes May be bound to a anti-TF antibodyeither directly, or indirectly by using an intermediary functionalgroup. Useful intermediary functional groups include chelators, such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid(see for instance U.S. Pat. No. 5,057,313),

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using anti-TF antibodies that are conjugated to dyes(such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or Molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, U.S. Pat.No. 6,331,175, which describes MRI techniques and the preparation ofantibodies conjugated to a MRI enhancing agent). Suchdiagnostic/detection agents may be selected from agents for use inmagnetic resonance imaging, and fluorescent compounds. In order to loadan anti-TF antibody with radioactive metals or paramagnetic ions, it maybe necessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail may be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which may bebound chelating groups such as, e.g., porphyrins, polyamines, crownethers, bisthiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose, Chelates may be coupled to anti-TF antibodiesusing standard chemistries.

Thus, the present invention provides diagnostic anti-TF antibodyconjugates, wherein the anti-TF antibody is conjugated to a contrastagent (such as for magnetic resonance imaging, computed tomography, orultrasound contrast-enhancing agent) or a radionuclide that may be, forexample, a gamma-, beta-, alpha-, Auger electron-, or positron-emittingisotope.

In a further aspect, the invention relates to a kit for detecting thepresence of TF antigen, or a cell expressing TF, in a sample comprising

an anti-TF antibody of the invention or a bispecific molecule of theinvention; and

instructions for use of the kit, wherein the kit in particular alsocontains an anti-TF antibody conjugated to a detection agent or contrastagent of the present invention.

In one embodiment, the anti-TF antibodies of the present invention mayalso be used in a kit for diagnosis of cancer comprising a containercomprising an anti-TF antibody, and one or more reagents for detectingbinding of the anti-TF antibody to a TF peptide, Such a kit may inparticular further comprise an anti-TF antibody drug conjugate of thepresent invention. Reagents may include, for example, fluorescent tags,enzymatic tags, or other detectable tags. The reagents may also includesecondary or tertiary antibodies or reagents for enzymatic reactions,wherein the enzymatic reactions produce a product that may bevisualized. In one embodiment, the present invention provides adiagnostic kit comprising one or more anti-TF antibodies, of the presentinvention in labeled or unlabeled form in suitable container(s),reagents for the incubations for an indirect assay, and substrates orderivatizing agents for detection in such an assay, depending on thenature of the label. Control reagent(s) and instructions for use alsomay be included.

Diagnostic kits may also be supplied for use with an anti-TF antibody,such as a conjugated/labeled anti-TF antibody, for the detection of acellular activity or for detecting the presence of TF peptides in atissue sample or host. In such diagnostic kits, as well as in kits fortherapeutic uses described elsewhere herein, an anti-TF antibodytypically may be provided in a lyophilized form in a container, eitheralone or in conjunction with additional antibodies specific for a targetcell or peptide. Typically, a pharmaceutical acceptable carrier (e.g.,an inert diluent) and/or components thereof, such as a Tris, phosphate,or carbonate buffer, stabilizers, preservatives, biocides, biocides,inert proteins, e.g., serum albumin, or the like, also are included(typically in a separate container for mixing) and additional reagents(also typically in separate container(s)). In certain kits, a secondaryantibody capable of binding to the anti-TF antibody, which typically ispresent in a separate container, is also included. The second antibodyis typically conjugated to a label and formulated in manner similar tothe anti-TF antibody of the present invention. Using the methodsdescribed above and elsewhere herein anti-TF antibodies may be used todefine subsets of cancer/tumor cells and characterize such cells andrelated tissues/growths.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledanti-TF antibodies (anti-TF antibody conjugated to a detection agent),of the present invention to such a specimen. The anti-TF antibody of thepresent invention may be provided by applying or by overlaying thelabeled anti-TF antibody of the present invention to a biologicalsample. Through the use of such a procedure, it is possible to determinenot only the presence of TF or TF-fragments but also the distribution ofsuch peptides in the examined tissue (e.g., in the context of assessingthe spread of cancer cells). Using the present invention, those ofordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) may be modified inorder to achieve such in situ detection.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Expression Constructs for Tissue Factor (TF)

Fully codon-optimized constructs for expression of TF or itsextracellular domains in HEK, NS0 or CHO cells, were generated. Theproteins encoded by these constructs are identical to Genbank accessionNP_(—)001984 for TF. The constructs contained suitable restriction sitesfor cloning and an optimal Kozak sequence (Kozak, 1987). The constructswere cloned in the mammalian expression vector pEE13.4 (Lonza Biologics)(Bebbington, Renner et al. 1992), obtaining pEE13.4TF. PCR was used toamplify the part, encoding the extracellular domain (ECD) (amino acid1-251) of TF, from the synthetic construct, adding a C-terminal His tagcontaining 6 His residues (TFECDHis). The construct was cloned inpEE13.4 and fully sequenced to confirm the correctness of the construct.

Example 2 Transient Expression in HEK-293F Cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, (HEK-293F)) cells were obtainedfrom Invitrogen and transfected with the appropriate plasmid DNA, using293fectin (Invitrogen) according to the manufacturer's instructions. Inthe case of antibody expression, the appropriate heavy chain and lightchain vectors, as described in Example 10, were co-expressed.

Example 3 Semi-Stable Expression in NS0 Cells

pEE13.4TF was stably transfected in NS0 cells and stable clones wereselected on growth in the absence of glutamine and in the presence of7.5 μM of methylsulphoximine (MSX). A pool of clones was grown insuspension culture while maintaining selection pressure. Pools weretested for TF expression by FACS analysis and secured for further use.

Example 4 Stable Expression in CH0 Cells

pEE13.4TF was stably transfected in CHO-K1SV (Lonza Biologics) cells andstable clones were selected on growth in the absence of glutamine and inthe presence of 50 μM MSX. Single clones were picked and expanded andtested for TF expression by FACS analysis as described below. Highexpressing clones were chosen and secured for further use.

Example 5

Purification of His-Tagged TF

TFECDhis was expressed I HEK-293F cells. The his-tag in TFECDHis enablespurification with immobilized metal affinity chromatography. In thisprocess, a chelator fixed onto the chromatographic resin is charged withCo²⁺ cations. TFECDHis-containing supernatant is incubated with theresin in batch mode (i.e. solution). The His-tagged protein bindsstrongly to the resin beads, while other proteins present in the culturesupernatant do not bind strongly. After incubation the beads areretrieved from the supernatant and packed into a column. The column iswashed in order to remove weakly bound proteins. The strongly boundTFECDHis proteins are then eluted with a buffer containing imidazole,which competes with the binding of His to Co²⁺. The eluent is removedfrom the protein by buffer exchange on a desalting column.

Example 6 Immunization Procedure of Transgenic Mice

Antibodies 042, 092-A09, 098 and 101 were derived from the followingimmunizations: three HCo20 mice (2 males and 1 female, strain GG2713),three HCo17 mice (2 males and 1 female, strain GG2714), threeHCo12-BALB/c mice (3 males, strain GG2811), three HCo7 (3 males, strainGG2201) and three HCo12 mice (3 males, strain GG2198) (Medarex, SanJosé, Calif., USA; for references see paragraph on HuMab mouse above)were immunized every fortnight alternating with 5×10⁶ semi-stabletransfected NS0-TF cells, or with 20 μg of TFECDHis protein. Eightimmunizations were performed in total, four intraperitoneal (IP) andfour subcutaneous (SC) immunizations at the tail base. The firstimmunization with cells was done in complete Freunds' adjuvant (CFA;Difco Laboratories, Detroit, Mich., USA), For all other immunizations,cells were injected IP in PBS and TFECDHis was injected SC usingincomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, Mich.,USA), When serum titers were found to be sufficient (dilution of serumof 1/50 or lower found positive in antigen specific screening assay asdescribed in example 7 on at least 2 sequential, biweekly screeningevents), mice were additionally boosted twice intravenously (IV) with 10μg TFECDHis protein in 100 μL PBS, 4 and 3 days before fusion.

Antibodies 109, 111 and 114 were derived from the followingimmunizations: three HCo20 mice (3 females), three HCo17 mice (3females), three HCo12-BALB/c mice (3 females), three HCo7 (3 males) andthree HCo12 mice (3 females) were immunized every fortnight with 5×10⁶semi-stable transfected NS0-TF cells. The first immunization with cellswas done in CFA, for all other (7) immunizations cells were injected IPin PBS. When serum titers were found to be sufficient (as definedabove), mice were additionally boosted twice IV with 1×10⁶ transientlysemi-stable transfected NS0-TF cells in 100 μL PBS, 4 and 3 days beforefusion.

Antibodies 011, 017-D12 and 025 were derived from the followingimmunizations: three HCo20 mice (3 males), three HCo17 mice (2 males and1 female), three HCo12-BALB/c mice (3 females), three HCo7 (3 males) andthree HCo12 mice (2 males and 1 female) were immunized every fortnightwith 20 μg of TFECDHis protein. The first (intraperitoneal) immunizationwith protein was done in CFA, for all other (7) immunizations proteinwas injected alternating subcutaneously and intraperitoneally in TFA.When serum titers were found to be sufficient (defined as above), micewere additionally boosted twice intravenously (IV) with 10 μg TFECDHisprotein in 100 μl PBS, 4 and 3 days before fusion.

Example 7 Homogeneous Antigen Specific Screening Assay

The presence of anti-TF antibodies in sera of immunized mice or HuMab(human monoclonal antibody) hybridoma or transfectoma culturesupernatant was determined by homogeneous antigen specific screeningassays (four quadrant) using Fluorometric Micro volume Assay Technology(FMAT; Applied Biosystems, Foster City, Calif., USA).

For this, a combination of 3 cell based assays and one bead based assaywas used. In the cell based assays, binding to TH1015-TF (HEK-293F cellstransiently expressing TF; produced as described above) and A431 (whichexpress TF at the cell surface) as well as HEK293 wild type cells (donot express TF, negative control) was determined. In the bead basedassay, binding to biotinylated TF coupled on a streptavidin bead(SB1015-TF) was determined.

Samples were added to the cells/beads to allow binding to TF.Subsequently, binding of HuMab was detected using a fluorescentconjugate (Goat anti-Human IgG-Cy5; Jackson ImmunoResearch). Mouseanti-human TF antibody (ERL; coupled to Alexa-647 at Genmab) was used aspositive control, HuMAb-mouse pooled serum and mouse-chrompure-Alexa647antibody were used as negative controls. The samples were scanned usingan Applied Biosystems 8200 Cellular Detection System (8200 CDS) and‘counts×fluorescence’ was used as read-out.

Example 8 HuMAb Hybridoma Generation

HuMab mice with sufficient antigen-specific titer development (definedas above) were euthanized and the spleen and lymph nodes flanking theabdominal aorta and vena cava were collected, Fusion of splenocytes andlymph node cells to a mouse myeloma cell line was done by electrofusionusing a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie,Md., USA), essentially according to the manufacturer's instructions.Selection and culturing of the resulting HuMab hybridomas was done basedupon standard protocols (e.g. as described in Coligan J. E., Bierer, B.E, Margulies, D. H., Shevach, E. M. and Strober, W., eds, CurrentProtocols in Immunology, John Wiley & Sons, Inc., 2006).

Example 9 Mass Spectrometry of Purified Antibodies

Small aliquots of 0.8 ml antibody containing supernatant from 6-well orHyperflask stage were purified using PhyTip columns containing Protein Gresin (PhyNexus Inc., San Jose, USA) on a Scicione ALH 3000 workstation(Caliper Lifesciences, Hopkinton, USA). The PhyTtip columns were usedaccording to manufacturers instructions, but buffers were replaced by:Binding Buffer PBS (B. Braun, Medical B. V., Oss, Netherlands) andElution Buffer 0.1M Glycine-HCl pH 2.7 (Fiuka Riedel-de Haën, Buchs,Germany). After purification, samples were neutralized with 2M Tris-HClpH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in somecases larger volumes of culture supernatant were purified using ProteinA affinity column chromatography.

After purification, the samples were placed in a 384-well plate (Waters,100 ul square well plate, part #186002631). Samples were deglycosylatedovernight at 37° C. with N-glycosidase F (Roche cat no 11365177001. DTT(15 mg/ml) was added (1 μl well) and incubated for 1 h at 37° C. Samples(5 or 6 ul) were desalted on an Acquity UPLC™ (Waters, Milford, USA)with a BEH300 C18, 1.7 μm, 2.1×50 mm column at 60° C. MQ water and LC-MSgrade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, TheNetherlands) with both 0.1% formic acid (Fluka, cat no 56302, Buchs,Germany), were used as Eluens A and B, respectively. Time-of-flightelectrospray ionization mass spectra were recorded on-line on amicrOTOFT™ mass spectrometer (Bruker, Bremen, Germany) operating in thepositive ion mode, Prior to analysis, a 900-3000 m/z scale wascalibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA).Mass spectra were deconvoluted with DataAnalysis™ software v. 3.4(Bruker) using the Maximal Entropy algorithm searching for molecularweights between 5 and 80 kDa.

After deconvolution the resulting heavy and light chain masses for allsamples were compared in order to find duplicate antibodies. In thecomparison of the heavy chains the possible presence of C-terminallysine variants was taken into account. This resulted in a list ofunique antibodies, where unique is defined as a unique combination ofheavy and light chains. In case duplicate antibodies were found, theresults from other tests were used to decide which was the best materialto continue experiments with.

MS analysis of the molecular weights of heavy and light chains of 118TF-specific hybridomas yielded 70 unique antibodies (unique heavychain/light chain combination). These were characterized in a number offunctional assays, identifying our lead candidates, TF specificantibodies.

Example 10 Sequence Analysis of the Anti-TF HuMAb Variable Domains andCloning in Expression Vectors

Total RNA of the anti-TF HuMabs was prepared from 5×10⁶ hybridoma cellsand 5′-RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA,using the SMART RACE cDNA Amplification kit (Clontech), according to themanufacturer's instructions. VH (variable region of heavy chain) and VL(variable region of light chain) coding regions were amplified by PCRand cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using the ZeroBlunt PCR cloning kit (Invitrogen). For each HuMab, 16 VL clones and 8VH clones were sequenced. The sequences are given in the SequenceListing and FIG. 1 herein.

Table 1A and Table 1B (below) give an overview of the antibody sequencesinformation and most homologous germline sequences.

TABLE 1A Heavy chain homologies V-GENE J-GENE D-GENE CDR-IMGT Ab andallele V-REGION Identity, % and allele and allele lengths 098IGHV1-69*04 95.49% (275/288 nt) IGHJ3*02 IGHD2-21*02 [8,8,11] 011IGHV3-23*01 96.53% (278/288 nt) IGHJ4*02 IGHD1-26*01 [8,8,11] 017IGHV3-23*01 98.26% (283/288 nt) IGHJ2*01 IGHD2-15*01 [8,8,13] 092IGHV3-23*01 97.92% (282/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,11] 101IGHV3-23*01 95.83% (276/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,11] 025IGHV3-30-3*01 97.57% (281/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,13] 109IGHV3-30-3*01 96.18% (277/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,13] 114IGHV3-33*01, or 94.44% (272/288 nt) IGHJ6*02 IGHD3-10*01 [8,8,12]IGHV3-33*03 111 IGHV3-30-3*01 97.57% (281/288 nt) IGHJ4*02 IGHD3-10*01[8,8,13] 042 IGHV3-23*01 98.26% (283/288 nt) IGHJ4*02 IGHD1-1*01[8.8.11]

TABLE 1B Light chain homologies CDR- V-GENE V-REGION J-GENE IMGT Ab andallele identity % ( nt) and allele lengths 011 IGKV1D-16*01 98.57%(275/279 nt) IGKJ2*01 [6.3.9] 092 IGKV1D-16*01 99.28% (277/279 nt)IGKJ2*01 [6.3.10] 098 IGKV1D-16*01 100.00% (279/279 nt) IGKJ2*01 [6.3.9]101 IGKV1D-16*01 100.00% (279/279 nt) IGKJ2*01 [6.3.10] 025 IGKV3-11*01100.00% (279/279 nt) IGKJ4*01 [6.3.9] 109 IGKV3-11*01 99.64% (278/279nt) IGKJ4*01 [6.3.9] 017 IGKV3-20*01 99.29% (280/282 nt) IGKJ1*01[7.3.9] 114 IGKV3-20*01 99.65% (281/282 nt) IGKJ4*01 [7.3.8] 111IGKV3-11*01 100.00% (279/279 nt) IGKJ4*01 [6.3.9] 042 IGKV3-20*01 99.29%(280/282 nt) IGKJ1*01 [7.3.9]

REFERENCES TO THE SEQUENCE LISTING Sequences in FIG. 1

In FIG. 1, the 017-D12 clone is referred to as “017” and similar the092-A09 clone is referred to as “092”,

VH-region SEQ ID No: 1 VH 114 SEQ ID No: 2 VH 114, CDR1 SEQ ID No: 3 VH114, CDR2 SEQ ID No: 4 VH 114, CDR3 SEQ ID No: 5 VH 011 SEQ ID No: 6 VH011, CDR1 SEQ ID No: 7 VH 011, CDR2 SEQ ID No: 8 VH 011, CDR3 SEQ ID No:9 VH 017-D12 SEQ ID No: 10 VH 017-D12, CDR1 SEQ ID No: 11 VH 017-D12,CDR2 SEQ ID No: 12 VH 017-D12, CDR3 SEQ ID No: 13 VH 042 SEQ ID No: 14VH 042, CDR1 SEQ ID No: 15 VH 042, CDR2 SEQ ID No: 16 VH 042, CDR3 SEQID No: 17 VH 092-A09 SEQ ID No: 18 VH 092-A09, CDR1 SEQ ID No: 19 VH092-A09, CDR2 SEQ ID No: 20 VH 092-A09, CDR3 SEQ ID No: 21 VH 101 SEQ IDNo: 22 VH 101, CDR1 SEQ ID No: 23 VH 101, CDR2 SEQ ID No: 24 VH 101,CDR3 SEQ ID No: 25 VH 025 SEQ ID No: 26 VH 025, CDR1 SEQ ID No: 27 VH025, CDR2 SEQ ID No: 28 VH 025, CDR3 SEQ ID No: 29 VH 109 SEQ ID No: 30VH 109, CDR1 SEQ ID No: 31 VH 109, CDR2 SEQ ID No: 32 VH 109, CDR3 SEQID No: 33 VH 098 SEQ ID No: 34 VH 098, CDR1 SEQ ID No: 35 VH 098, CDR2SEQ ID No: 36 VH 098, CDR3 SEQ ID No: 37 VH 111 SEQ ID No: 38 VH 111,CDR1 SEQ ID No: 39 VH 111, CDR2 SEQ ID No: 40 VH 111, CDR3 VL-region SEQID No: 41 VL 114 SEQ ID No: 42 VL 114, CDR1 SEQ ID No: 43 VL 114, CDR2SEQ ID No: 44 VL 114, CDR3 SEQ ID No: 45 VL 011 SEQ ID No: 46 VL 011,CDR1 SEQ ID No: 47 VL 011, CDR2 SEQ ID No: 48 VL 011, CDR3 SEQ ID No: 49VL 017-D12 SEQ ID No: 50 VL 017-D12, CDR1 SEQ ID No: 51 VL 017-D12, CDR2SEQ ID No: 52 VL 017-D12, CDR3 SEQ ID No: 53 VL 042 SEQ ID No: 54 VL042, CDR1 SEQ ID No: 55 VL 042, CDR2 SEQ ID No: 56 VL 042, CDR3 SEQ IDNo: 57 VL 092-A09 SEQ ID No: 58 VL 092-A09, CDR1 SEQ ID No: 59 VL092-A09, CDR2 SEQ ID No: 60 VL 092-A09, CDR3 SEQ ID No: 61 VL 101 SEQ IDNo: 62 VL 101, CDR1 SEQ ID No: 63 VL 101, CDR2 SEQ ID No: 64 VL 101,CDR3 SEQ ID No: 65 VL 025 SEQ ID No: 66 VL 025, CDR1 SEQ ID No: 67 VL025, CDR2 SEQ ID No: 68 VL 025, CDR3 SEQ ID No: 69 VL 109 SEQ ID No: 70VL 109, CDR1 SEQ ID No: 71 VL 109, CDR2 SEQ ID No: 72 VL 109, CDR3 SEQID No: 73 VL 098 SEQ ID No: 74 VL 098, CDR1 SEQ ID No: 75 VL 098, CDR2SEQ ID No: 76 VL 098, CDR3 SEQ ID No: 77 VL 111 SEQ ID No: 78 VL 111,CDR1 SEQ ID No: 79 VL 111, CDR2 SEQ ID No: 80 VL 111, CDR3Anti-TF HuMab 092-A09 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:17 and the VL sequenceof SEQ ID No: 57.Anti-TF HuMab 101 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:21 and the VL sequenceof SEQ ID No: 61.Anti-TF HuMab 025 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:25 and the VL sequenceof SEQ ID No: 65.Anti-TF HuMab 109 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:29 and the VL sequenceof SEQ ID No: 69.Anti-TF HuMab 017-D12 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:9 and the VL sequenceof SEQ ID No: 49.Anti-TF HuMab 114 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:1 and the VL sequenceof SEQ ID No: 41.Anti-TF HuMab 042 is a full length, fully human monoclonal IgG1,κantibody comprising the VH sequence of SEQ ID No:13 and the VL sequenceof SEQ ID No: 53.Anti-TF HuMab 011 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:5 and the VL sequenceof SEQ ID No: 45.Anti-TF HuMab 098 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:33 and the VL sequenceof SEQ ID No: 73.Anti-TF HuMab 111 is a full length, fully human monoclonal IgG1, κantibody comprising the VH sequence of SEQ ID No:37 and the VL sequenceof SEQ ID No: 77.

Example 11 Purification of Antibodies

Culture supernatant was filtered over 0.2 μm dead-end filters and loadedon 5 ml Protein A columns (rProtein A FF, Amersham Bioscience) andeluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediatelyneutralized with 2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mMNaH₂PO₄, 140 mM NaCl, pH 7.4 (B. Braun). After dialysis samples weresterile filtered over 0.2 μm dead-end filters. Purity was determined bySDS-PAGE and concentration was measured by nephelometry and absorbanceat 280 nm. Purified antibodies were aliquoted and stored at −80° C. Oncethawed, purified antibody aliquots were kept at 4° C. Mass spectrometrywas performed to identify the molecular mass of the antibody heavy andlight chains expressed by the hybridomas as described in Example 9.

Example 12 Binding of Anti-TF HuMabs to the Extracellular Domain of TFin ELISA

The specificity of the obtained anti-TF HuMabs was evaluated by ELISA.ELISA plates (Microlon; Greiner Bio-One) were coated overnight at +4° C.with 0.5 μg/mL of TFECDHis in PBS, pH 7.4. Coated ELISA plates wereemptied and blocked for one hour at room temperature with 2% (v/v)chicken serum (Gibco, Paisley, Scotland) in PBS and washed with PBScontaining 0.05% Tween 20 (PBST). Subsequently, HuMabs, serially dilutedin PBSTC (PBS supplemented with 2% (v/v) chicken serum and 0.05% (v/v)Tween-20), were incubated for 1 hr at RT under shaking conditions (300rpm). Bound HuMabs were detected using HRP-conjugated goat-anti-humanIgG antibodies (Jackson ImmunoResearch) diluted 1:5,000 in PBSTC, whichwere incubated for 1 hr at RT under shaking conditions (300 rpm). Thereaction was further developed with ABTS (Roche Diagnostics) at RT inthe dark, stopped after 15-30 minutes by adding 2% (w/v) oxalic acid andthen the absorbance at 405 nm was measured. HuMab-KLH (a humanmonoclonal antibody against KLH (keyhole limpet haemocyanin)), was usedas a negative control. Mouse anti-human TF (ERL) was used as positivecontrol (HRP labeled anti-mouse IgG as conjugate). Binding curves wereanalyzed using non-linear regression (sigmoidal dose-response withvariable slope) using GraphPad Prism V4.03 software.

As can been seen in FIG. 3, all of the anti-TF antibodies boundTFECDHis. The EC₅₀ values for the HuMabs are the mean of 3 experimentsand varied between 0.13 and 0.17 nM (Table 2 below).

TABLE 2 HuMab TF EC₅₀ nM 11 0.16 017-D12 0.25 42 0.23 092-A09 0.18 1010.28 98 0.13 114 0.17 25 0.34 109 0.27

Example 13 Binding of Anti-TF HuMabs to Membrane-Bound TF

Binding of anti-TF HuMabs to membrane-bound TF was determined by FACSanalysis, using TF transfected CHO cells, or TF expressing tumor celllines MDA-MB-231, (luciferase transfected) A431 and Bx-PC3.

Cells were resuspended in PBS (2×10⁶ cells/mL), put in 96-well V-bottomplates (50 μL/well). 50 μL of serially diluted HuMab in FACS buffer (PBSsupplemented with 0.1% BSA and 0.02% Na-azide) was added to the cellsand incubated for 30 minutes on ice. After washing three times with FACSbuffer, 50 μL of phycoerythrin (PE)-conjugated goat anti-human IgGFc(Jackson ImmunoResearch), diluted 1:100 in FACS buffer, was added. After30 minutes on ice (in the dark), cells were washed three times, andspecific binding of the HuMabs was detected by flow cytometry on aFACSCalibur (BD Biosciences). HuMab-KLH was used as a negative control.Mouse anti-TF followed by PE-conjugated anti-mouse IgGFc was used aspositive control. Binding curves were analyzed using non-linearregression (sigmoidal dose-response with variable slope) using GraphPadPrism V4.03 software (GraphPad Software, San Diego, Calif., USA).

FIG. 4 shows an example of binding curves of TF-specific HuMabs toMDA-MB-231 cells. Table 3 gives an overview of EC₅₀ values of binding ofTF-specific HuMabs to TF transfected CH0 cells (S1015-TF), MDA-MB-231,A431 and Bx-PC3 cells.

TABLE 3 Overview of EC₅₀ and maximum mean fluorescence intensity (maxMFI) values determined by FACS analysis of binding of TF-specific HuMabsto different cell types. MDA-MB-231 Bx-PC3 A431 S1015-TF-012 group HuMabTF EC₅₀ Max MFI EC₅₀ Max MFI EC₅₀ Max MFI EC₅₀ Max MFI I 13 1.58 24511.86 1305 8.04 3622 1.07 5207 I 44 0.87 1881 1.88 1136 1.45 2646 2.135021 I 87-Lg6 8.28 1107 7.19 1030 nt nt nt nt II 11 0.47 2143 1.01 12800.20 2606 1.32 5654 II 017-D12 1.33 2401 1.61 1422 1.24 3296 1.21 5792II 42 0.25 1518 2.45 1701 nt nt nt nt II 092-A09 0.53 2290 0.84 12620.83 3137 1.32 5409 II 101 0.85 2071 2.25 1220 3.16 2934 1.77 5859II/III 98 0.99 1956 1.38 1151 1.40 2755 0.96 5229 II/III 114 0.47 24380.80 1407 0.90 3433 1.72 6095 III 3 3.20 1798 4.98 1106 6.94 2530 2.064247 III 25 0.69 2254 0.88 1320 5.19 3170 0.73 5808 III 109 2.16 20524.04 1324 1.74 3124 0.92 5629 III 111 1.03 1774 1.83 1128 2.88 3043 0.555353

EC₅₀ values are in nM. Max MFI for MDA-MB-231, BxPC3 and A431 cells at30 μg/mL antibody, for S1015-TF at 7.5 μg/mL antibody.

Example 14 Inhibition of FVIIa Binding to TF

Inhibition of binding of FVIIa to TF, on MDA-MB-231 cells, by anti-TFHuMabs was measured by FACS analysis. MDA-MB-231 cells were washed inPBS to remove serum and plated in 96-well plates (100,000 cells perwell). Cells were incubated with anti-TF HuMabs in DMEM/0.1% BSA for 15min, followed by incubation with 100 nM FVIIa in DMEM/0.1% BSA at 4° C.for 30 min. Cells were washed with PBS/0.1% BSA/0.02% sodium azide (FACSbuffer) and incubated with 10 μg/mL rabbit anti-FVIIa (Abcam [ab7053])).Cells were washed with FACS buffer and incubated with 1:50 dilutedPE-labeled goat anti-rabbit IgG (Jackson [111-116-144]). Cells werewashed with FACS buffer and mean fluorescence intensity (MFI) wasmeasured on a FACSCanto II (Becton Dickinson).

The concentration of antibody needed to obtain 50% inhibition (IC₅₀) wascalculated using GraphPad Prism (non-linear regression analysis).

FIG. 5 and Table 4 shows that HuMab-TF-098 (IC₅₀: 1.2 μg/mL), -114 (IC₅₀could not be determined) and -011 (IC₅₀: 0.6 μg/mL) efficientlyinhibited FVIIa binding to MDA-MB-231 cells, while HuMab-TF-013, -044and -111 did not (or to a much lesser extent) inhibit FVIIa binding.

TABLE 4 overview of IC₅₀ values of anti-TF-HuMabs to inhibit FVIIabinding. Antibody (HuMab-TF-) IC₅₀ 098 1.218 111 ND^(a)) 013 ND^(a)) 044ND^(a)) 114 ND^(a)) 011  0.6472 ^(a))could not be calculated

Data shown are IC₅₀ values (in μg/mL) of anti-TF HuMabs to inhibitbinding of 100 nM FVIIa to TF on MDA-MB-231 cells, measured in onerepresentative experiment.

Example 15 Antibody-mediated Internalization and Cell Killing by Anti-TFHuMabs in a Kappa-ETA′Assay

To determine if anti-TF HuMabs are suitable for an antibody-drugconjugate approach, a generic in vitro cell-based killing assay usingkappa-directed pseudomonas-exotoxin A (anti-kappa-ETA′) was used. Inthis assay a high affinity anti-human kappa light chain domainconjugated to a truncated form of the pseudomonas-exotoxin A was used.Upon internalization, the anti-kappa-ETA′ domain-antibody conjugateundergoes proteolysis and disulfide-bond reduction, separating thecatalytic and the binding domain. The catalytic domain is transportedfrom the Golgi system to the endoplasmic reticulum via the KDELretention motif, and subsequently translocated to the cytosol where itinhibits protein synthesis and induces apoptosis (Kreitman R J BioDrugs.2009; 23(1): 1-13. Recombinant Immunotoxins Containing TruncatedBacterial Toxins for the Treatment of Hematologic Malignancies).

Antibody-mediated internalization and cell killing by the toxin wastested for different anti-TF HuMabs. Three different cell lines, withcomparable levels of TF expression, were tested. These cells alsoexpressed EGFR (at different levels), allowing the use of a positivecontrol antibody (2F8), that binds EGFR and is known to induce EGFRinternalization. The number of TF and EGFR molecules expressed on thecell lines was determined by Qifi kit (Dako, (Glostrup, Denmark); A431cells: average TF molecules per cell approximately 500,000, average EGFRmolecules per cell approximately 500,000; BxPC3: average TF moleculesper cell approximately 500,000, average EGFR molecules per cellapproximately 200,000; MDA-MB-231: average TF molecules per cellapproximately 500,000, average EGFR molecules per cell approximately100,000. Cells were seeded in optimal concentration (A431: 2,500cells/well; BxPC3: 3,000 cells/well; MDA-MB-231: 5,000 cells/well) incell culture medium in 96-well tissue culture plates (Greiner Bio-one)and allowed to adhere. To identify anti-TF HuMabs that enableinternalization of and killing by the toxin, a fixed concentration (0.5μg/mL. [A431 and BxPC3]; 0.25 μg/mL [MDA-MB-231]) of anti-kappa-ETA′,that did not induce non-specific cell death in the absence of antibody,was incubated for 30 min with a titrated amount of anti-TF HuMabs beforeaddition to the cells. After three days, the amount of viable cells wasquantified with AlamarBlue (BioSource International, San Francisco, US),according to the manufacturer's instructions. Fluorescence was monitoredusing the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland)with standard AlamarBlue settings. 2F8 with the anti-kappa-ETA′ wasincluded as a positive control. An isotype control antibody (IgG1-b12)was used as negative control.

FIG. 6 and Table 5 show that all but one (HuMab-TF-087) of theanti-kappa-ETA′-pre-incubated anti-TF HuMabs were able to kill A431,BxPC3 and MDA-MB-231 cells in a dose-dependent manner.Anti-kappa-ETA′-pre-incubated HuMab-TF-098, -114 and -011, induced moreefficient killing (EC₅₀ between 9×10⁻⁵ and 4×10⁻⁴ μg/mL on A431 cells)than anti-kappa-ETA′-pre-incubated HuMab-TF-013, -111 and -044 (EC₅₀between 2.0×10⁻² and 9.8×10⁻² μg/mL on A431 cells).Anti-kappa-ETA′-pre-incubated HuMab-TF-087 did not induce cell killing.

One representative experiment is shown for each cell line: A431 (a),BxPC3 (b) and MDA-MB-231 (c). Data shown are mean fluorescenceintensities (MFI) ±S.E.M. of triplicate wells of cells treated withanti-kappa-ETA′-pre-incubated anti-TF HuMabs. The upper dashed lineindicates the maximal signal obtained in the absence ofanti-kappa-ETA′-pre-incubated anti-TF HuMabs; the lower dashed lineindicates maximal killing obtained with staurosporine.

TABLE 5 overview of EC₅₀ values and percentages of cell killing inducedby anti-kappa-ETA′-pre-incubated anti-TF-HuMabs. A431 BxPC3 MDA-MB-231Antibody EC₅₀ EC₅₀ EC₅₀ (HuMab-TF-) % kill μg/mL % kill μg/mL % killμg/mL 098 95 9.0 × 10⁻⁵ 99 1.3 × 10⁻⁵ 96 7.2 × 10⁻⁴ 111 92 3.4 × 10⁻² 981.5 × 10⁻² 88 2.3 × 10⁻² 013 80 2.0 × 10⁻² 96 9.4 × 10⁻³ 56 N.D.^(a) 04466 9.8 × 10⁻² 96 1.5 × 10⁻² 44 N.D.^(a) 087 3 N.D.^(a) 56 N.D.^(a) 8N.D.^(a) 114 97 2.6 × 10⁻⁴ 99 7.3 × 10⁻⁴ 99 2.5 × 10⁻³ 011 96 3.9 × 10⁻⁴98 2.6 × 10⁻⁴ 88 3.0 × 10⁻³ 2F8 99 7.1 × 10⁻⁶ 98 3.5 × 10⁻⁵ 84 1.5 ×10⁻³ B12 5 N.D.^(a) 22 N.D.^(a) 0 N.D.^(a) ^(a)Could not be calculated.

Data shown are EC₅₀ values (in μg/mL) and maximal percentages kill ofthe indicated cell lines treated with anti-kappa-ETA′-pre-incubatedanti-TF HuMabs, measured in one representative experiment, Percentage ofcell killing (% kill) was calculated as follows;MFI_(untreated)−MFI_(conjugated HuMab-treated))/(MFI_(untreat)−MFI_(stauroporine-treated)).

Example 16 Preparation of anti-TF ADCs

HuMab-011, HuMab-098 and HuMab-111 and the negative control IgG1-b12were produced transiently in HEK-293F cells (HuMab-011, HuMab-111 andIgG1-b12) or using a stable CHO cell line (HuMab-098). The antibodieswere purified by Protein A chromatography according to standardprocedures, finally yielding approximately 400 mg of purified antibody.Next, the antibodies were conjugated to vcMMAE and mcMMAF, respectively.Approximately 200 mg of HuMab-011, HuMab-098 or HuMab-111 was conjugatedto either vcMMAE or mcMMAF. The drug-linker vcMMAE or mcMMAF wasalkylated to the cysteines of the reduced antibodies according toprocedures described in the literature (Sun et al. (2005) BioconjugateChem. 16: 1282-1290; McDonagh et (2006) Protein Eng. Design Sel. 19:299-307; Alley et al., (2008) Bioconjugate Chem. 19: 759-765). Thereaction was quenched by the addition of an excess of N-acetylcysteine.Any residual unconjugated drug was removed by purification and the finalanti-TF antibody drug conjugates were formulated in PBS. The anti-TFantibody drug conjugates were subsequently analyzed for concentration(by absorbance at 280 nm), the drug to antibody ratio (the ‘DAR’) byreverse phase chromatography (RP-HPLC) and hydrophobic interactionchromatography (HIC), the amount of unconjugated drug (by reverse phasechromatography), the percentage aggregation (by size-exclusionchromatography, SEC-HPLC) and the endotoxin levels (by LAL). The resultsare shown below in table 6.

TABLE 6 overview of different characteristics of the antibody-drugconjugates HuMab-TF-011 HuMab-TF-098 HuMab-TF-111 IgG1-b12 Assay vcMMAEmcMMAF vcMMAE mcMMAF vcMMAE mcMMAF vcMMAE mcMMAF Concentration 10.699.86 9.28 10.96 9.83 10.4 5.49 8.74 (mg/mL) DAR by RP- 3.9 3.9 3.9 4.04.3 4.1 3.6 3.9 HPLC DAR by HIC 3.9 4.1 3.7 3.9 4.1 4.2 3.4 3.9 %unconjugated <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 drug % aggregate5.3 5.3 0.8 0.7 1.2 0.8 0.6 1.0 by SEC-HPLC Endotoxin 0.2 0.2 0.131<0.05 0.07 0.07 0.05 <0.05

Example 17 Binding of the Anti-TF ADCs to Recombinant ExtracellularDomain of TF, Determined by ELISA

Binding of the anti-TF ADCs to TF was measured by ELISA (coatedrecombinant extracellular domain of TF) and compared with binding ofunconjugated anti-TF HuMabs. ELISA plates (Greiner BioOne) were coatedO/N at 4° C. with 1.25 μg/mL, 100 μL per well, recombinant TFECDHis inPBS (B. Braun Melsungen AG). ELISA plates were washed three times withPBS containing 0.05% Tween-20 (PBST), blocked with 200 μL/well PBST atRT for 1 h while shaking (300 rpm), washed three times with PBST andemptied. Subsequently, 100 μL anti-TF ADCs or unconjugated anti-TFHuMabs were added in serial dilutions in PBST and incubated whileshaking at RT for 90 min. ELISA plates were washed three times with PBSTand emptied. Bound anti-TF ADCs and unconjugated HuMabs were detected byadding HRP-conjugated mouse-anti human IgG1 (100 μL; 0.015 μg/mL;Sanquin; # M1328) in assay buffer and incubation while shaking at RT for1 h. Plates were washed three times with PBST, emptied and incubatedwith 100 μL ABTS solution (50 ml ABTS buffer [Roche] and one ABTS tablet[50 mg; Roche]). After incubation in the dark at RT for 30 min, thereaction was stopped by incubation with 100 μL per well oxalic acid (2%[w/v]; Riedel de Haen) in the dark, for 10 min, Plates were measured atOD 405 nm in an ELISA reader (Biotek Instruments, EL808 AbsorbanceMicroplate Reader).

IgG1-b12, an antibody binding to a non-related antigen, was used as anegative control (both unconjugated as well as in ADC format).

Binding curves were analyzed by non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism 5 software(GraphPad Software, San Diego, Calif., USA).

FIG. 7 shows binding curves, demonstrating that all the anti-TF HuMabsand ADCs bound within a similar range to the TF extracellular domain inan ELISA (EC₅₀ values between 370 and 470 ng/mL).

Table 7 shows EC₅₀ values of anti-TF HuMabs and ADCs for binding to theextracellular domain of TF. The EC₅₀ values are in ng/mL.

TABLE 7 Overview of EC₅₀ values for binding of TF-specific HuMabs andADCs to the extracellular domain of TF, determined by ELISA. EC₅₀(ELISA) HuMab-TF- Unconjugated vcMMAE mcMMAF 011 373 469 431 098 422 426401 111 377 464 416

Example 18 Antibody-Mediated Internalization and Cell Killing by Anti-TFADCs in an In Vitro Killing Assay

To determine the capacity of anti-TF ADCs to induce cytotoxicity, an invitro cell-based killing assay was performed.

Cell killing of three cell lines was tested for different anti-TF ADCs.A431 cells were obtained from Deutsche Sammlung von Mikroorganimsmen undZellkulturen GmbH (DSMZ: ACC 91), HPAF-II and NCI-H441 cells wereobtained from the American Type Culture Collection (ATCC: CRL-1997 andHTB-174). Cells were seeded in optimal concentration (A431: 2.5×10³cells/well; HPAF-II and NCI-H441: 5×10³ cells/well) in cell culturemedium in 96-well tissue culture plates (Greiner Bio-one) and allowed toadhere. Serial dilutions of anti-TF ADCs were added and incubated at 37°C. for 72 h (A431 and HPAF-II) or 96 h (NCI-H441). The amount of viablecells was quantified with AlamarBlue (BioSource International, SanFrancisco, US), according to the manufacturer's instructions.Fluorescence was monitored using the EnVision 2101 Multilabel reader(PerkinElmer, Turku, Finland) with standard AlamarBlue settings.IgG1-b12 (an antibody binding to a non-related antigen) ADCs were usedas negative controls, Staurosporine (Sigma, # S6942) was used to inducemaximal cell killing.

The A431 and HPAF-II cell lines both express more than 200,000 tissuefactor molecules per cell and may therefore be regarded as expressinghigh levels of tissue factor.

NCI-H441 cells express approximately 80,000 tissue factor molecules percell and may therefore be regarded as expressing intermediate levels oftissue factor.

FIG. 8 and Table 8 show that all anti-TF ADCs were able to kill A431,HPAF-II and NCI-H441 cells in a dose-dependent manner, HuMab-TF-098 and-011 induced slightly more efficient killing (IC₅₀ between 9 and 22ng/mL on A431 cells, between 1 and 5 ng/mL on HPAF-II cells and between1 and 10 ng/mL on NCI-H441 cells) than HuMab-TF-111 (IC₅₀ between 46 and83 ng/mL on A431 cells, between 4 and 15 ng/mL on HPAF-II cells and 416ng/mL on NCI-H441 cells). One representative experiment is shown foreach cell line: A431 (a) and HPAF-II (b). Data shown are percentagessurvival ±S.E.M. of duplicate wells of cells treated with anti-TF ADCs.

TABLE 8 overview of IC₅₀ values and percentages of cell killing inducedby anti-TF ADCs. ADC A431 HPAF-II NCI-H441 (HuMab-TF-) % kill IC₅₀ %kill IC₅₀ % kill IC₅₀ 098-vcMMAE 92 9 71 1 60 10 098-mcMMAF 85 13 73 563 4 011-vcMMAE 93 10 71 3 60 10 011-mcMMAF 78 22 72 5 53 5 111-vcMMAE90 46 73 4 51 416 111-mcMMAF 73 83 74 15 62 416 IgG1-b12-vcMMAE 0N.D.^(a)) 0 N.D.^(a)) 0 N.D.^(a)) IgG1-b12-mcMMAF 0 N.D.^(a)) 0N.D.^(a)) 0 N.D.^(a)) ^(a))Could not be calculated.

Data shown are IC₅₀ values (in ng/mL) and maximal percentages kill (at aconcentration of 10 μg/mL) of the indicated cell lines treated withanti-TF ADCs, measured in one representative experiment. Percentage ofcell killing (% kill) was calculated as follows:(MFI_(untreated)−MFI_(anti-TF ADC-treated))/(MFI_(untreated)−MFI_(stauroporine-treated))×100%.

Example 19 Therapeutic Treatment of A431 and HPAF-II Tumor XenograftsSCID Mice with Anti-TF ADCs

The in vivo efficacy of anti-TF ADCs was determined in establishedsubcutaneous (SC) A431 and HPAF-II xenograft tumors in SOD mice. 5×10⁶A431 (obtained from DSMZ) or 2×10⁶ HPAF-II (obtained from ATCC) tumorcells in 200 μL PBS were injected SC in the right flank of female SCIDmice, followed by four injections with anti-TF ADCs or controls(IgG1-b12; both as ADC and unconjugated), starting when tumor sizes wereapproximately 200-250 mm³ for A431 xenografts: day 11, day 14, day 18and day 21 or approximately 100-150 mm³ for HPAF-II xenografts: day 13,16, 20 and 23 (60 μg/mouse in 100 μL, intraperitoneally (IP)). Tumorvolume was determined at least two times per week. Tumor volumes (mm³)were calculated from caliper (PLEXX) measurements as:0.52×(length)×(width)².

FIG. 9 shows that all anti-TF ADCs were effective in inhibiting tumorgrowth of established s.c. A431 (a) and HPAF-II (b) tumors. The datashown are mean tumor volumes S.E.M. per group (n=7 mice per group). Inthe HPAF-II model, vcMMAE conjugates were significantly more efficientin inhibiting tumor growth than mcMMAF conjugates.

Example 20 Stability of Anti-TF Lead Clone ADCs and IgG3-b12 ADCs

The stability of the MMAE- and MMAF-conjugated materials was tested uponstorage for 10 days, 1, 2 and 3 months at <−65° C. and 5° C. In thisexample only the three months data are shown, since similar results wereobtained for all intermediate time points. Furthermore, the stability ofthe materials was tested upon repeated cycles of freeze-thawing.

Prepared ADC batches (four IgG batches each conjugated with twodifferent linkers, Table 6 were deep frozen. For stability testing,batches were thawed and diluted to 1 mg/mL in PBS. The diluted materialwas aliquoted into 300 μL portions in cryovials and vials were placed at<−65° C. or 5° C. for temperature storage. For freeze-thawing, threevials of each batch were frozen at <−65° C., 0/N, and then thawedunassisted at RT. The freeze-thaw cycle was repeated another two times(the samples were freeze-thawed three times in total). All materialswere analyzed at the start of the study (t=0) by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), High Performance SizeExclusion Chromatography (HP-SEC) and binding to tissue factor(TFECDHis) in a binding ELISA. The same analyses were performed forsamples stored for three months (t=3 months) at <65° C. and 5° C. andfor freeze-thaw samples.

SDS-PAGE was performed under reducing and non-reducing conditions on4-12% NuPAGE Bis-Tris gels (Invitrogen, Breda, The Netherlands) using amodified Laemli method (Laemli 1970 Nature 227(5259): 680-5), where thesamples were run at neutral pH. The SDS-PAGE gels were stained withCoomassie and digitally imaged using an Optigo Imaging System (IsogenLife Science).

HP-SEC was performed using a Waters Affiance 2695 or 2795 separationunit (Waters, Etten-Leur, The Netherlands) connected to a TSK HP-SECcolumn (G3000SWxl; Tosoh Bioscience, via Omnilabo, Breda, TheNetherlands) and a Waters 2487 dual λ absorbance detector (Waters).Samples were run at 1 mL/min, Results were processed using Empowersoftware version 2 and expressed per peak as percentage of total peakheight.

Binding to recombinant protein of the TF extracellular domain wasanalyzed by ELISA, as described supra in example 17.

FIG. 10 a-d show SDS-PAGE analyses of unconjugated and conjugatedanti-TF lead clones and IgG1-b12 at the start of the stability study(t=0). On non-reducing SOS-PAGE (a,b), unconjugated IgG1 migrated as anintact IgG band of about 150 kDa. As expected, ADCs largely dissociatedinto IgG fragments of smaller sizes (125 kDa=HHL, 99 kDa=HH, 67 kDa=HL,51 kDa=H and 25 kDa=L), due to the denaturing SDS-PAGE conditions andthe non-covalent nature of the ADC molecules (disrupted disulphidebonds) (FIG. 10 a,b), Reduced SDS-PAGE analysis (FIG. 10 c,d) showedbands of the unconjugated light chain (L0) and light chain with one drug(MMAE or MMAF) attached (L1). Partial resolution was observed for theunconjugated heavy chain (H0) and the MMAE-conjugated forms (H1, H2 andH3). The MMAF-conjugated and unconjugated heavy chain forms could not bewell resolved but appeared as a diffuse band at 50 kDa.

The SOS-PAGE results for the samples after three months storage at bothtemperatures (<−65° C. and 5° C.) were comparable to the t=0 data, asshown in FIG. 10 e-f. Also for the freeze-thaw samples, no differenceswere observed compared with the start material by SDS-PAGE analysis(data not shown).

FIG. 11 shows the HP-SEC profile overlays for the ADC batches at t=0 andt=3 months at both temperatures. Under native HP-SEC conditions, ADCmaterial (t=0) eluted as one peak of monomeric IgG molecules with minoramounts of dimeric IgG molecules. No changes were observed for the MMAE-and MMAF-conjugated HuMab-TF-098 (a, b) and HuMab-TF-011 (c, d) uponthree months storage. However, ADC material of HuMab-TF-111 (e, f) andIgG1-b12 (g, h) showed a decrease in recovery (peak height) at t=3months. This lower recovery was already observed in the t=10 dayssamples and remained constant after prolonged storage up to threemonths.

The percentage of monomeric IgG molecules (% monomer) was calculatedfrom the HP-SEC peak profile and the data are summarized in Table 9. Forcomparison, the % monomer of unconjugated material is shown. The datashow that >95% of the ADC material consisted of intact monomeric IgGmolecules. The % monomer remained unchanged after three months storageat <−65° C. and 5° C., indicating that no aggregates were formed intime.

HP-SEC analysis of the freeze/thaw samples showed that IgG peak recoveryof all samples was similar to recoveries at t=0 (data not shown).However, freeze-thawing of the HuMab-TF-ADC material resulted in aslightly lower % monomer (1.5-3.6%), as shown in Table 9. This was dueto the formation of minor amounts of aggregates (dimeric IgG moleculesas judged by HP-SEC, data not shown).

Binding of unconjugated and conjugated HuMab-TF-098, -011 and -111 torecombinant protein of the TF extracellular domain (TFECDHis) was testedby ELISA. After three months storage at <−65° C. and 5° C., the bindingcapacity did not change compared with that at t=0, as shown in FIG. 12.Similar results were obtained for the freeze-thaw samples (data notshown).

The stability experiments show that the ADC material, at 1 mg/mL, wasstable at <−65° C. and at 5° C. for at least three months, as determinedby SDS-PAGE, HP-SEC and binding to TFECDHis. Minor aggregate formationwas induced by repeated freeze thawing of the material.

TABLE 9 HP-SEC analysis of ADC samples. Data shown are percentagesmonomeric molecules. freeze-thaw t = 3 months (3 separate vials)linker-toxin T = 0 <−65° C. 5° C. 1 2 3 HuMab-TF-098 unconjugated >99 —— — — — vcMMAE 98.3 97.6 98.3 96.8 96.1 96.2 mcMMAF 95.4 98.2 98.2 92.392.0 91.9 HuMab-TF-011 unconjugated 96.1 — — — — — vcMMAE 96.3 95.2 95.693.4 93.0 92.9 mcMMAF 95.8 96.6 96.4 94.2 93.5 93.7 HuMab-TF-111unconjugated >99 — — — — — vcMMAE 98.3 98.3 98.4 96.5 94.6 95.9 mcMMAF97.9 97.8 >99 95.5 95.1 94.8 IgG1-b12 unconjugated >99 — — — — — vcMMAE98.2 96.2 97.3 98.3 98.2 98.3 mcMMAF 98.6 98.8 98.8 98.1 97.9 98.0

Example 21 Dose-Response of Anti-TF ADCs in Therapeutic Treatment ofHPAF-II Tumor Xenografts in SCID Mice

The in vivo efficacy of anti-TF ADCs was further analyzed by treatmentof established SC HPAF-II xenograft tumors in SCID mice with differentdoses of anti-TF ADCs. HPAF-II tumor xenografts were established asdescribed supra, followed by four injections with anti-TF vcMMAE ADCs intwo different doses (6 and 20 μg/mouse [IgG1-b12 was added to a finaldose of 60 μg IgG1 per mouse] in 100 μL, IP) or control unconjugated mAb(IgG1-b12; 60 μg/mouse in 100 μL, IP); starting when tumor sizes wereapproximately 100-150 mm³: day 10, 13, 17 and 21. Tumor volume wasdetermined at least two times per week. Tumor volumes (mm³) werecalculated from caliper (PLEXX) measurements as: 0.52×(length)×(width)².

FIG. 13 shows that the 20 μg doses of all three vcMMAE conjugates wereeffective in inhibiting tumor growth of established s.c. HPAF-II tumors.The 6 μg dose of all three vcMMAE conjugates was capable of slightlydelaying, but not inhibiting tumor growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. Any combination ofthe embodiments disclosed in the dependent claims are also contemplatedto be within the scope of the invention

The invention claimed is:
 1. An antibody drug conjugate comprising anantibody comprising a heavy chain variable region (VH) and a light chainvariable region (VL), wherein: (i) the VH region comprises a CDR1comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3comprising the amino acid sequence set forth in SEQ ID NO: 8, and the VLregion comprises a CDR1 comprising the amino acid sequence set forth inSEQ ID NO: 46, a CDR2 comprising the amino acid sequence set forth inSEQ ID NO: 47, and a CDR3 comprising the amino acid sequence set forthin SEQ ID NO: 48, (ii) the VH region comprises a CDR1 comprising theamino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 36, and the VL regioncomprises a CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 74, a CDR2 comprising the amino acid sequence set forth in SEQ IDNO: 75, and a CDR3 comprising the amino acid sequence set forth in SEQID NO: 76, (iii) the VH region comprises a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 38, a CDR2 comprising the aminoacid sequence set forth in SEQ ID NO: 39, and a CDR3 comprising theamino acid sequence set forth in SEQ ID NO: 40, and the VL regioncomprises a CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 78, a CDR2 comprising the amino acid sequence set forth in SEQ IDNO: 79, and a CDR3 comprising the amino acid sequence set forth in SEQID NO: 80, or (iv) the VH region comprises a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the aminoacid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the aminoacid sequence set forth in SEQ ID NO: 4, and the VL region comprises aCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, aCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, anda CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44,wherein the antibody has been conjugated to an auristatin or afunctional peptide analog or derivate thereof via a linker.
 2. Anantibody drug conjugate comprising an antibody which binds to tissuefactor and comprises heavy and light chain variable regions, wherein:(i) the VH region comprises the amino acid sequence of SEQ ID NO: 5 orthe VL region comprises the amino acid sequence of SEQ ID NO: 45, (ii)the VH region comprises the amino acid sequence of SEQ ID NO: 33 or theVL region comprises the amino acid sequence of SEQ ID NO: 73, (iii) theVH region comprises the amino acid sequence of SEQ ID NO: 37 or the VLregion comprises the amino acid sequence of SEQ ID NO: 77, or (iv) theVH region comprises the amino acid sequence of SEQ ID NO: 1 or the VLregion comprises the amino acid sequence of SEQ ID NO:
 41. 3. Theantibody drug conjugate according to claim 1, wherein the antibody is afull length antibody.
 4. The antibody drug conjugate according to claim1, wherein the antibody is a human monoclonal IgG1 antibody.
 5. Theantibody drug conjugate according to claim 1, wherein the auristatin ismonomethyl auristatin E (MMAE):


6. The antibody drug conjugate according to claim 1, wherein theauristatin is monomethyl auristatin F (MMAF):


7. The antibody drug conjugate according to claim 1, wherein the linkeris attached to sulphydryl residues of the antibody obtained by partialreduction of the antibody.
 8. The antibody drug conjugate according toclaim 1, wherein the linker-auristatin is vcMMAF or vcMMAE:

wherein p denotes a number of from 1 to 8, S represents a sulphydrylresidue of the antibody, and Ab designates the antibody.
 9. The antibodydrug conjugate according to claim 1, wherein the linker-conjugate ismcMMAF:

wherein p denotes a number of from 1 to 8, S represents a sulphydrylresidue of the antibody, and Ab designates the antibody.
 10. Acomposition comprising the antibody drug conjugate of claim 1 and apharmaceutically acceptable carrier.
 11. The antibody drug conjugateaccording to claim 2, wherein the antibody comprises: (i) a VH regioncomprising the amino acid sequence of SEQ ID NO: 5 and a VL regioncomprising an amino acid sequence of SEQ ID NO: 45, (ii) a VH regioncomprising the amino acid sequence of SEQ ID NO: 33 and a VL regioncomprising an amino acid sequence of SEQ ID NO: 73, (iii) a VH regioncomprising the amino acid sequence of SEQ ID NO: 37 and a VL regioncomprising an amino acid sequence of SEQ ID NO: 77, or (iv) a VH regioncomprising the amino acid sequence of SEQ ID NO: 1 and a VL regioncomprising an amino acid sequence of SEQ ID NO: 41.